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Rat Model of Chronic Midthoracic Lateral Hemisection
大鼠慢性胸部中段脊髓测半切模型   

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Abstract

Although most spinal cord injuries (SCI) are anatomically incomplete, only limited functional recovery has been observed in people and rats with partial lesions. To address why surviving fibers cannot mediate more complete recovery, it is important to evaluate the physiological and anatomical status of spared fibers. These experiments require use of animal models. Here we describe a midthoracic unilateral spinal cord hemisection (HX; corresponds to Brown-Sequard lesion in humans) in adult rats. This is a useful animal model for partial injuries because there is a clear lesion of one entire side of the cord with intact fibers remaining on the contralateral side. This model allows the study and comparison of how acute and chronic trauma affect function of the surviving fibers.

Materials and Reagents

  1. Adult (~210 g) female Sprague-Dawley rats
  2. 1.5% isoflurane
  3. Heated workstation with gas evacuation system and a face mask for induction and maintenance of anesthesia (ProStation Kit) (MIP/Anesthesia Technology, catalog number: AS-01-0491 )
  4. Antibiotic (Baytril)
  5. Analgesic (Buprenorphine)
  6. Sterile lactated Ringer solution
  7. Cotton swap
  8. Buprenorphine
  9. Petrolatum ophthalmic ointment (Dechra Veterinary Products)
  10. 4-0 monocryl (Ethicon)
  11. Wound clips
  12. Anatomical tracers

Equipment

  1. Isoflurane induction chamber (1 L)
  2. Water circulating heating pad
  3. Surgical microscope
  4. Iridectomy scissors, faucets, blades, other tools for small animal surgery

Procedure

Note: All procedures were performed on adult female Sprague Dawley rats (~200 g) in compliance with the Institutional Animal Care and Use Committee at SUNY-Stony Brook and Northport VAMC.

  1. After pre-training on the behavioral tasks, rats were deeply anesthetized with 3% isoflurane in 100% O2 in an induction chamber (1 L).
  2. Anesthesia was maintained by administering 1.5% isoflurane in 100% O2 through a face mask. A water circulating heating pad was used to maintain body temperature at 36.5-37 °C during surgeries.
  3. Before surgery, animals received a subcutaneous injection of analgesic Buprenorphine (0.01 mg/kg) to reduce post-operative pain. Petrolatum ophthalmic ointment was applied to the eyes to prevent desiccation.
  4. Dorsal laminectomy (i.e. partial vertebral laminectomy) was performed to expose T10 spinal segment. The spinal level was confirmed by using a vein at T5-T6 as a landmark. The meningeal layer at T10 was slit (1 mm) at the midline longitudinally.
  5. A complete transection of the left hemicord at T10 was carried out with the tip of iridectomy scissors, as follows:
    1. First, while holding the dura and lifting the spinal cord slightly, one tip of the scissors was passed through the entire thickness of the spinal cord dorsal to ventral at the midline;
    2. The left dorsal and ventral columns were then cut from lateral to the midline by closing other tip of scissors;
    3. Finally, while keeping the cord elevated, one tip of the scissors was placed under the ventral surface of the spinal cord (up to the midline) and any uncut tissue in the left dorsal and ventral columns was cut ventral to dorsal up to the midline.


      Figure 1. Images of spinal cord following dorsal laminectomy and lateral HX lesion of the spinal cord. A. Image of the exposed spinal cord (at arrow) following a dorsal laminectomy procedure. B. Image of rat brain and spinal cord isolated from the rat that recived HX spinal cord injury (at arrow) 6 weeks erlier.

  6. After surgery, the muscles were closed with 4-0 monocryl suture and skin was closed with wound clips.
  7. Antibiotic (5 mg/kg, sc) and 5 ml of lactated Ringer’s solution were administered subcutaneously. Bladder function was not compromised by this surgery. Injections of antibiotic, analgesic and Ringer`s solution were administered for 3 days post injury.
  8. Horizontal or transverse sections of the spinal cord were used for reconstruction of injury (Figure 2).


    Figure 2. Lateral HX spinal cord injury. A. Horizontal section of the rat spinal cord prepared immediately after HX. B. Transverse section of the cord at SCI epicenter prepared 6 weeks after HX; highlighted is area of spared white matter. Scale bar, 100 μm. (Adopted and modified from Garcia-Alias et al., 2011).

  9. As a result of HX SCI, there are clear behavioral impairments revealed by challenging motor tasks and automated Catwalk gait analysis; electrophysiological experiments allow evaluation of the conduction through fibers contralateral to the lesion and the possibility of establishing a functional detour around the lesion following administration of various treatments; moreover, unilateral injections of the anatomical tracers permit visualization of anerogradely labeled midline crossing fibers and retrogradely labeled neurons (Arvanian et al., 2009; Hunanyan et al., 2010; Schnell et al., 2011; Garcia-Alias et al., 2011; Hunanyan et al., 2011; Petrosyan et al., 2013).

Acknowledgments

This protocol was adapted from previously published papers: Arvanian et al. (2009); Hunanyan et al. (2011); García-Alías et al. (2011); Schnell et al. (2011). The research was supported by Merit Review Funding from the Department of Veterans Affairs and the Department of Defense and New York State Spinal Cord Injury Research Board.

References

  1. Arvanian, V. L., Schnell, L., Lou, L., Golshani, R., Hunanyan, A., Ghosh, A., Pearse, D. D., Robinson, J. K., Schwab, M. E., Fawcett, J. W. and Mendell, L. M. (2009). Chronic spinal hemisection in rats induces a progressive decline in transmission in uninjured fibers to motoneurons. Exp Neurol 216(2): 471-480.
  2. Garcia-Alias, G., Petrosyan, H. A., Schnell, L., Horner, P. J., Bowers, W. J., Mendell, L. M., Fawcett, J. W. and Arvanian, V. L. (2011). Chondroitinase ABC combined with neurotrophin NT-3 secretion and NR2D expression promotes axonal plasticity and functional recovery in rats with lateral hemisection of the spinal cord. J Neurosci 31(49): 17788-17799.
  3. Hunanyan, A. S., Garcia-Alias, G., Alessi, V., Levine, J. M., Fawcett, J. W., Mendell, L. M. and Arvanian, V. L. (2010). Role of chondroitin sulfate proteoglycans in axonal conduction in mammalian spinal cord. J Neurosci 30(23): 7761-7769.
  4. Hunanyan, A. S., Alessi, V., Patel, S., Pearse, D. D., Matthews, G. and Arvanian, V. L. (2011). Alterations of action potentials and the localization of Nav1.6 sodium channels in spared axons after hemisection injury of the spinal cord in adult rats. J Neurophysiol 105(3): 1033-1044.
  5. Petrosyan, H. A., Hunanyan, A. S., Alessi, V., Schnell, L., Levine, J. and Arvanian, V. L. (2013). Neutralization of inhibitory molecule NG2 improves synaptic transmission, retrograde transport, and locomotor function after spinal cord injury in adult rats. J Neurosci 33(9): 4032-4043.
  6. Schnell, L., Hunanyan, A. S., Bowers, W. J., Horner, P. J., Federoff, H. J., Gullo, M., Schwab, M. E., Mendell, L. M. and Arvanian, V. L. (2011). Combined delivery of Nogo-A antibody, neurotrophin-3 and the NMDA-NR2d subunit establishes a functional 'detour' in the hemisected spinal cord. Eur J Neurosci 34(8): 1256-1267.

简介

虽然大多数脊髓损伤(SCI)在解剖学上是不完全的,但是在具有部分损伤的人和大鼠中仅观察到有限的功能恢复。 为了解决为什么存活的纤维不能介导更完全的恢复,重要的是评估备用纤维的生理和解剖状态。 这些实验需要使用动物模型。 在这里我们描述成人大鼠的midthoracic单侧脊髓半切(HX;对应于人类中的棕色Sequard病变)。 这是用于部分损伤的有用的动物模型,因为在完整纤维保留在对侧的情况下,存在帘线的整个侧面的清楚的病变。 该模型允许研究和比较急性和慢性创伤如何影响存活纤维的功能。

材料和试剂

  1. 成年(〜210g)雌性Sprague-Dawley大鼠
  2. 1.5%异氟烷
  3. 带有排气系统的加热工作站和用于感应和维持麻醉的面罩(ProStation Kit)(MIP /麻醉科技,目录号:AS-01-0491)
  4. 抗生素(Baytril)
  5. 镇痛药(丁丙诺啡)
  6. 无菌乳酸林格氏溶液
  7. 棉花交换
  8. 丁丙诺啡
  9. 凡士林眼膏(Dechra Veterinary Products)
  10. 4-0单烯丙基(Ethicon)
  11. 伤口夹
  12. 解剖示踪剂

设备

  1. 异氟烷诱导室(1L)
  2. 水循环加热垫
  3. 手术显微镜
  4. 铱切割剪刀,水龙头,刀片,其他小动物手术工具

程序

注意:根据在SUNY-Stony Brook和Northport VAMC的Institutional Animal Care and Use Committee,对成年雌性Sprague Dawley大鼠(〜200g)进行所有程序。

  1. 在对行为任务进行预训练后,在诱导室(1L)中用3%异氟烷在100%O 2中深度麻醉大鼠。
  2. 通过经由面罩施用在100%O 2中的1.5%异氟烷维持麻醉。在手术期间,使用水循环加热垫来维持体温在36.5-37℃
  3. 在手术前,动物接受皮下注射镇痛药丁丙诺啡(0.01mg/kg)以减少手术后疼痛。将凡士林眼用软膏施用于眼睛以防止干燥。
  4. 进行背侧椎板切除术(即椎间盘切除术)以暴露T10脊柱节段。通过使用T5-T6处的静脉作为标志物来确认脊柱水平。将T10处的膜层切开(1 mm)。
  5. 使用虹膜切除术剪刀的尖端在T10处完全横断左半球,如下:
    1. 首先,在保持硬脊膜和略微抬起脊髓的同时,将剪刀的一个尖端穿过背脊脊髓的整个厚度至中线处的腹侧;
    2. 然后通过关闭剪刀的其他尖端从中线的横向切割左背侧和右侧柱;
    3. 最后,在保持绳索升高的同时,将剪刀的一个尖端放置在脊髓的腹侧表面下方(直到中线),并且将左侧背侧和腹侧柱中的任何未切割的组织切成腹侧至背侧至中线。


      图1.脊髓背侧椎板切除术和脊髓侧HX病变的图像。 A.背侧椎板切除术后暴露脊髓的图像(箭头)。 B.从大鼠分离的大鼠脑和脊髓的图像,所述大鼠在6周前收集HX脊髓损伤(箭头)。

  6. 手术后,用4-0单缝缝合闭合肌肉,用伤口夹闭皮肤。
  7. 皮下施用抗生素(5mg/kg,皮下)和5ml乳酸林格氏溶液。膀胱功能没有受到这种手术。在损伤后给予抗生素,止痛剂和林格氏溶液的注射3天。
  8. 脊髓的水平或横向切片用于损伤的重建(图2)。


    图2.横向HX脊髓损伤 A.在HX后立即制备的大鼠脊髓的水平切片。 B.在HX后6周制备的SCI震中线的横截面;突出显示的是幸免白质的面积。比例尺,100μm。 (由Garcia-Alias等人于2011年采纳和修改)。

  9. 作为HX SCI的结果,通过挑战运动任务和自动步行步态分析显示出明显的行为障碍;电生理学实验允许评估通过对着损伤的纤维的传导以及在施用各种治疗之后在损伤周围建立功能性迂回的可能性;此外,解剖示踪剂的单侧注射允许可视化的航空标记的中线交叉纤维和逆行标记的神经元(Arvanian等人,2009; Hunanyan等人,2010; Schnell 2011; Garcia-Alias等人,2011; Hunanyan等人,2011; Petrosyan等人,2011年; ,2013)。

致谢

该协议改编自以前发表的论文:Arvanian等人(2009); Hunanyan等人(2011); García-Alías等人(2011); Schnell等人(2011)。该研究得到退伍军人事务部,国防部和纽约州脊髓损伤研究委员会的优秀评审资助。

参考文献

  1. Arvanian,VL,Schnell,L.,Lou,L.,Golshani,R.,Hunanyan,A.,Ghosh,A.,Pearse,DD,Robinson,JK,Schwab,ME,Fawcett,JWand Mendell, )。 大鼠的慢性脊髓半切除术诱导未损伤的纤维向运动神经元的传递的进行性下降。 Exp Neurol em> 216(2):471-480。
  2. Garcia-Alias,G.,Petrosyan,H.A.,Schnell,L.,Horner,P.J.,Bowers,W.J.,Mendell,L.M.,Fawcett,J.W.and Arvanian,V.L。 软骨素酶ABC结合神经营养因子NT-3分泌和NR2D表达促进大鼠的轴突可塑性和功能恢复侧脑半切的脊髓。 31(49):17788-17799。
  3. Hunanyan,A.S.,Garcia-Alias,G.,Alessi,V.,Levine,J.M.,Fawcett,J.W.,Mendell,L.M.and Arvanian,V.L.(2010)。 硫酸软骨素蛋白多糖在哺乳动物脊髓轴突传导中的作用 Neurosci 30(23):7761-7769。
  4. Hunanyan,A.S.,Alessi,V.,Patel,S.,Pearse,D.D.,Matthews,G.and Arvanian,V.L。(2011)。 动脉电位的改变和Nav1.6钠通道在罕见轴突中的局部化脊髓在成年大鼠中的作用。 105(3):1033-1044。
  5. Petrosyan,H.A.,Hunanyan,A.S.,Alessi,V.,Schnell,L.,Levine,J.and Arvanian,V.L。(2013)。 抑制性分子NG2的中和改善成年脊髓损伤后的突触传递,逆行转运和运动功能大鼠。 J Neurosci 33(9):4032-4043。
  6. Schnell,L.,Hunanyan,A.S.,Bowers,W.J.,Horner,P.J.,Federoff,H.J.,Gullo,M.,Schwab,M.E.,Mendell,L.M.and Arvanian,V.L。 Nogo-A抗体,神经营养因子-3和NMDA-NR2d亚基的组合递送建立了功能性" in the hemisected spinal cord。 Eur J Neurosci 34(8):1256-1267。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Arvanian (former Arvanov), V. (2013). Rat Model of Chronic Midthoracic Lateral Hemisection. Bio-protocol 3(18): e912. DOI: 10.21769/BioProtoc.912.
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