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Contusion Spinal Cord Injury Rat Model
挫伤脊髓损伤大鼠模型   

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Abstract

Spinal cord injury (SCI) can lead to severe disability, paralysis, neurological deficits and even death. In humans, most spinal cord injuries are caused by transient compression or contusion of the spinal cord associated with motor vehicle accidents. Animal models of contusion mimic the typical SCI’s found in humans and these models are key to the discovery of progressive secondary tissue damage, demyelination, and apoptosis as well as pathophysiological mechanisms post SCI. Here we describe a method for the establishment of an efficient and reproducible contusion model of SCI in adult rat.

Keywords: Spinal cord injury(脊髓损伤), Contusion(挫伤), Rat(大鼠), Demyelination(脱髓鞘)

Background

The spinal cord plays an important role in the interconnections between the brain and peripheral nerves. Severe SCI causes the loss of physiological functions and even paralysis or death (Singh et al., 2014). After SCI, the microvascular hemorrhage with disruption of the blood-spinal cord barrier is followed by edema, ischemia, and the release of cytotoxic chemicals from inflammatory pathways (Oyinbo, 2011; Mothe and Tator, 2012). Secondary neurodegenerative events such as demyelination, Wallerian degeneration and axonal dieback occur in the non-permissive tissue environment. Contusion, a type of blunt injury in the spinal cord, mimics typical SCI in humans which is mainly caused by vehicle accidents, especially motorcycles. In contrast to the sharp SCI model such as the transection that provides an anatomical model for evaluating axonal regeneration, the contused spinal cord presents a preferable microenvironment for studying of pathophysiological mechanisms post injury (Young, 2002). Experimental induction of a contusive SCI in a rat model using the NYU-MASCIS (New York University-Multicenter Animal Spinal Cord Injury Study) impactor device has been validated as an analog to human SCI. Furthermore, a comparison between the rat model of SCI with human SCI shows functional electrophysiological and morphological evidence of similar patterns recorded in motor evoked potentials and somatosensory evoked potentials (SSEP) as well as high-resolution magnetic resonance imaging (Basso et al., 1996; Metz et al., 2000; Kwon et al., 2002; Young, 2002). Here we describe a method with tips for construction of an efficient and reproducible contusion model of SCI in adult rat.

Materials and Reagents

  1. Surgical blade #21 (DIMEDA Instrumente, catalog number: 06.121.00 )
  2. Chromic catgut (4/0) (UNIK, catalog number: CT134 )
  3. Nylon suture (3/0) (UNIK, catalog number: NC203 )
  4. Adult female Sprague Dawley (SD) rat (225-250 g)
  5. Isoflurane (Halocarbon Laboratories, NDC12164-002-25 )
  6. 0.9% saline solution (TAI YU CHEMICAL & PHARMACEUTICAL, catalog number: RH1704 )
  7. Povidone-iodine solution (YING YUAN CHEMICAL PHARMACEUTICAL, catalog number: S-166 )
  8. Acetaminophen solution (CENTER Laboratories, catalog number: 19746 )
  9. Luxol fast blue stain kit (Abcam, catalog number: ab150675 )
  10. Hematoxylin and Eosin Stain Kit (Vector Laboratories, catalog number: H-3502 )
  11. Trimethoprim-sulfamethoxazole pre-mixed antibacterial solution (YUNG SHIN PHARM, catalog number: TRI-004 )
  12. Trimethoprim-sulfamethoxazole antibacterial injectable working solution (see Recipes)

Equipment

  1. NYU-MASCIS weight-drop impactor with an alligator and the software
  2. 2.5 mm tip of impactor for rat
  3. Scalpel handle #4 (DIMEDA Instrumente, catalog number: 06.104.00 )
  4. Heating pad
  5. Adson toothed forceps (DIMEDA Instrumente, catalog number: 10.180.12 )
  6. ALM self-retaining retractor (DIMEDA Instrumente, catalog number: 18.620.07 )
  7. MAYO HEGAR needleholder (DIMEDA Instrumente, catalog number: 24.180.16 )
  8. Littauer bone cutter (Stoelting, catalog number: 52167-80P )
  9. Operating scissors (Shinetech, catalog number: ST-S114PK )
  10. CMA/150 Temperature controller (CMA Microdialysis, model: CMA 150 , catalog number: 600)
  11. Dry sterilizer (Braintree Scientific, model: Germinator 500 , catalog number: GER 5287-120V)
  12. Surgical microscope (Carl Zeiss, model: Zeiss Stativ S3 )
  13. Table top anesthesia system (AM Bickford, catalog number: 61020 )
  14. EVA soft foam mat (Lee Chyun Enterprise, model: FM 600T )

Software

  1. MAS 7.0 version
  2. Microsoft Windows 98 operating system

Procedure

Ethical statement: Adult female Sprague-Dawley (SD) rats (225-250 g) were used in this protocol. All procedures involving animals were approved by the Animals Committee of Taipei Veterans General Hospital (permit numbers IACUC 2014-137 and IACUC 2015-253) and were in accordance with the Guide for the Care and Use of Laboratory Animals outlined by the National Institutes of Health.

  1. All instruments (toothed forceps, tip, clamps, retractor, needle holder, scalpel handle and scissors) that touch the inside of the wound must be sterilized using the dry sterilizer.
  2. Set up the PC and start the impactor program.
  3. Place the rat into the induction chamber.
  4. Turn on the airflow (1-1.5 L/min with isoflurane 5%) and then monitor the rat until recumbent.
  5. Move the anesthetized rat from the chamber to the mask of anesthesia system and set the level of isoflurane to 1.5-2%.
  6. Maintain the rat’s core body temperature at 36-37 °C on a warming pad with an electrical temperature controller of the rectal probe.
  7. Shave the thoracic area and apply the povidone-iodine solution on the shaved area.
  8. Use the scalpel to make a longitudinal incision on the dorsal thoracic surface and dissect the paraspinal muscle to expose the vertebrae T7-T12.
    Note: The spinous process of T2 is longer than any of the others. Touch the position of T2 under the skin to determine the approximate position for operation with your finger (Figure 1A).
  9. Use the retractor to gently pull the paravertebral muscles away from the spines. The laminectomy is performed with a Zeiss operating microscope (under 7.5x magnification). Cut and remove the bones of T8, T9 and partial T10 with the bone cutter and toothed forceps. This will expose the dorsal surface of the spinal cord without disrupting the dura. (Figure 1B)
    Note: To avoid hitting the vertebral bone with the tip of impactor, the bone must be removed to obtain more than 2.5 mm width because the diameter of the impactor tip is 2.5 mm.
  10. Two stabilization clamps are used to immobilize the posterior spinous processes of the vertebrae T7 and T12 and to support the vertebral column during contusion.
  11. Place the rat with the center of T8-T10 spinal cord under the tip (Figures 1C). The ground alligator of the impactor is placed on the muscle to form an electrical conductance between the tip and the spinal cord.
    Note: Using the toothed forceps push the middle of the clamped vertebral bone slightly in order to ensure the vertebral bone is clamped well and is stable (Figure 1D).
  12. Place the tip at 0 mm and then lower down the rod to let the tip touch the dura of the spinal cord (Figure 1E, Left). When the tip touches the dura, the black box of the impactor will produce light and make a buzzing sound (Figure 1E, Right).


    Figure 1. Laminectomy, impact site and animal care. A. The approximate positions of the 2nd thoracic spine (T2, black circle) and the 9th thoracic spine (white circle) shown on the dorsal thoracic surface. B. The spinal cord has been exposed by a T8-T10 laminectomy before contusion (white circles are vertebral locations). C. The rat placed at the center of the rod and the clamps was immobilized to show the spinous processes of the vertebrae T7 and T12. D. The toothed forceps pushed the middle of clamped vertebral bone slightly in order to ensure the vertebral bone is clamped well and stable. E. The tip was placed at 0 mm by an inserted pin; the tip can be raised up and held at specific heights (6.25, 12.5, 25 or 50 mm; Left). When the tip touches the dura, the black box of the impactor will produce light and buzzing sound (Right). F. The site of spinal cord with subdural hemorrhage on the T9 vertebral position after contused. G. The muscle was continuously closed with chromic catgut (Left), and the skin was interrupted suturing with nylon suture (Right). H. Holding the rat with one hand and gently squeezing the bladder on the lower abdomen with the thumb and first two fingers of the other hand.

  13. Raise the rod and hold the tip by the inserted pin at specific heights (included 6.25, 12.5, 25 or 50 mm). Manually pull the inserted pin to let the rod fall onto the exposed dura by gravity at T9 to produce a contusion injury.
    Note: The parameters for the impactor that can be recorded by the software are: impact velocity (Vi), cord compression distance (Cd), time (Ct), and rate (Cr = Cd/Ct). For instance, the impact velocity from a 50 mm height should be achieved in 0.98 m/sec. The compression distance should be around 2-3 mm by software display, if it is greater than 3 mm, that means the spinal cord is not clamped well. The Ct presents the time required for the rod to compress the spinal cord to the deepest point.
  14. After contused, the subdural hemorrhage can be seen clearly under dura (Figure 1F).
  15. Raise the tip and release the clamps gently from the vertebral column.
  16. The wound is continuously closed with chromic catgut (4/0) for muscle, and interrupted suturing with nylon suture (3/0) for skin (Figure 1G).
  17. Turn off the anesthesia system and then place the rat back to the cage which is on the heating pad.
    Note: The entire procedure takes about 1 h, and the rat should be woken up within 10-20 min after removing the isoflurane. Slightly press the paw of the rat, the reflex action of hind limb should not be present 24 h after SCI. In our experience, the mortality rate of rat is under 5%.
  18. Monitor the rat and provide post-operative care by daily observation for signs of distress including weight loss, dehydration and bladder dysfunction. Provide 3 ml of sterile 0.9% saline subcutaneously for rehydration. Prophylactic antibiotics (Trimethoprim-sulfamethoxazole antibacterial injectable solution, 1:30 diluted in 0.9% normal saline, 2 ml/kg) are injected subcutaneously daily in 5 days post-surgery. The analgesic acetaminophen (65 mg/kg) is orally delivered if the rat shows the sign of self-mutilation. In addition, if the rat shows serious foot damage, we usually discard the rat.
  19. The rat is taken care of in a conventional animal house and maintained at a 12-h light-dark cycle. Manual emptying of the rat’s bladder is performed twice daily by squeezing the lower abdomen (Figure 1H).
    Note: Most rats show hematuria at 1-3 days after contused. If bloody urine does not empty well or a large residual volume of urine is left in the bladder after SCI, it might cause cystitis, infection, and even death.

Data analysis

  1. Lesion volume
    SCI resulted in cavitation and demyelination, which expanded the extent of damage (Poon et al., 2007). Luxol fast blue (LFB) and hematoxylin and eosin (H&E) stains were used to identify cavities and myelinated white matter respectively. The staining procedures followed the manufacturer’s protocols. Images were photographed from the rostral end to caudal end throughout the injury site at 2.5x magnification with a microscope camera. Contusion (50-mm height) caused the most of gray matter losing and few white matter sparing (Figure 2).


    Figure 2. Histological staining at 6th week after SCI. One of the representative results showed that SCI rat by LFB staining (Upper) and the continued slides for H&E staining (Bottom) (-2 mm, 0 mm, and 2 mm; scale bars = 250 μm).

  2. Locomotor score
    The Basso, Beattie, and Bresnahan (BBB) open field score is used to evaluate locomotion of the hindlimbs (Basso et al., 1996). Briefly, the rat was placed on the mat (size: 100 x 100 x 40 mm) and scored by BBB test from 0 (no observable hindlimb movement) to 21 (normal hind-limb movement) points. The low end of the BBB score (0-8) is characterized by each hind-limb joint movements, the intermediate (9-14) and high (15-21) are characterized by weight support, forelimb hindlimb coordination stepping, toe clearance, predominant paw position and tail position (for a detailed description of BBB scores, please see the reference by Basso et al., 1996). Behavioral analyses were conducted and recorded using a video camera every week by both blinded examiners. The weekly scores of each hindlimb from examiners were averaged together to yield one score. Groups by different grades (or treatments) could be compared using a two-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. According to the study by Basso et al. (1996), the locomotor scores were greatest in the 6.25 mm group and lowest in the 50 mm group (please refer to step 13). The 6.25 mm group demonstrated the maximal functional recovery to near the normal locomotion within 3 weeks after contused. The 12.5 mm group recovered quickly from no or slight hind-limb joint movements to consistently stepping within 3 weeks. In contrast, the 25 mm group presented slower improvement of locomotion after contused. The 50 mm group showed paralysis within 3-7 days and no weight-support recovery in the following 6 weeks. In addition, Mestre et al. (2015) and our previous study yielded similar results (Video 1) in 50 mm group of SD rat at 6th week after contused (Chiu et al., 2016).

    Video 1. Locomotor recovery in 50 mm group. The rat showed paralysis within 1-7 days and no weight-support recovery in the following 6 weeks.

Recipes

  1. Trimethoprim-sulfamethoxazole antibacterial injectable working solution
    Trimethoprim-sulfamethoxazole, 1 vol
    Antibacterial solution diluted in 0.9% normal saline, 30 vol

Acknowledgments

This study was supported by the postdoctoral fellows program of Academia Sinica, grants of Ministry of Science and Technology, Taiwan (MOST 104-2314-B-010-012-MY3), and Taipei Veterans General Hospital (105V-E6-001-MY3-1). We also thank the Dr. May-Jywan Tsai for her help and Neural Regeneration Laboratory of Taipei Veterans General Hospital for providing experimental space and facilities.

References

  1. Basso, D. M., Beattie, M. S. and Bresnahan, J. C. (1996). Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection. Exp Neurol 139(2): 244-256.
  2. Chiu, C. W., Huang, W. H., Lin, S. J., Tsai, M. J., Ma, H., Hsieh, S. L. and Cheng, H. (2016). The immunomodulator decoy receptor 3 improves locomotor functional recovery after spinal cord injury. J Neuroinflammation 13(1): 154.
  3. Kwon, B. K., Oxland, T. R. and Tetzlaff, W. (2002). Animal models used in spinal cord regeneration research. Spine 27(14): 1504-1510.
  4. Mestre, H., Ramirez, M., Garcia, E., Martiñón, S., Cruz, Y., Campos, M. G. and Ibarra, A. (2015). Lewis, Fischer 344, and sprague-dawley rats display differences in lipid peroxidation, motor recovery, and rubrospinal tract preservation after spinal cord injury. Front Neurol 6: 108.
  5. Metz, G. A., Curt, A., van de Meent, H., Klusman, I., Schwab, M. E. and Dietz, V. (2000). Validation of the weight-drop contusion model in rats: a comparative study of human spinal cord injury. J Neurotrauma 17(1): 1-17.
  6. Mothe, A. J. and Tator, C. H. (2012). Advances in stem cell therapy for spinal cord injury. J Clin Invest 122(11): 3824-3834.
  7. Oyinbo, C. A. (2011). Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade. Acta Neurobiol Exp (Wars) 71(2): 281-299.
  8. Poon, P. C., Gupta, D., Shoichet, M. S. and Tator, C. H. (2007). Clip compression model is useful for thoracic spinal cord injuries: histologic and functional correlates. Spine 32: 2853-9.
  9. Singh, A., Tetreault, L., Kalsi-Ryan, S., Nouri, A. and Fehlings, M. G. (2014). Global prevalence and incidence of traumatic spinal cord injury. Clin Epidemiol 6: 309-331.
  10. Young, W. (2002). Spinal cord contusion models. Prog Brain Res 137: 231-255.

简介

脊髓损伤(SCI)可导致严重的残疾,麻痹,神经功能障碍甚至死亡。 在人类中,大多数脊髓损伤是由与机动车辆事故相关的脊髓的短暂压迫或挫伤引起的。 痉挛的动物模型模仿人类发现的典型SCI,这些模型是发现继发性继发性组织损伤,脱髓鞘和凋亡以及SCI后的病理生理机制的关键。 这里我们描述了一种在成年大鼠中建立SCI有效和可重复的挫伤模型的方法。
【背景】脊髓在大脑和周围神经之间的互连中起重要作用。严重的SCI导致生理功能丧失甚至瘫痪或死亡(Singh等,2014)。 SCI后,伴随血脊髓屏障破裂的微血管出血随后发生水肿,局部缺血和细胞毒性化学物质从炎症途径的释放(Oyinbo,2011; Mothe和Tator,2012)。继发性神经变性事件如脱髓鞘,Wallerian变性和轴索缺血发生在非允许组织环境中。挫伤是脊髓中的一种钝性损伤,模拟人类的典型SCI,主要是由车辆事故造成的,特别是摩托车。与提供用于评估轴突再生的解剖模型的横断面的尖锐SCI模型相反,挫伤的脊髓是用于研究损伤后病理生理机制的优选微环境(Young,2002)。使用NYU-MASCIS(纽约大学 - 多中心动物脊髓损伤研究)冲击器装置的大鼠模型中的痉挛性SCI的实验诱导已经被验证为与人类SCI的类似物。此外,SCI与人类SCI的大鼠模型之间的比较显示了在运动诱发电位和躯体感觉诱发电位(SSEP)以及高分辨率磁共振成像中记录的类似模式的功能电生理和形态学证据(Basso等,1996 ; Metz等,2000; Kwon等,2002; Young,2002)。在这里,我们描述了一种方法,其中提供了用于构建成年大鼠SCI的高效和可重复的挫伤模型的提示。

关键字:脊髓损伤, 挫伤, 大鼠, 脱髓鞘

材料和试剂

  1. 外科刀片#21(DIMEDA Instrumente,目录号:06.121.00)
  2. 铬肠(4/0)(UNIK,目录号:CT134)
  3. 尼龙缝线(3/0)(UNIK,目录号:NC203)
  4. 成年女性Sprague Dawley(SD)大鼠(225-250克)
  5. 异氟烷(Halocarbon Laboratories,NDC12164-002-25)
  6. 0.9%盐水溶液(TAI YU CHEMICAL& PHARMACEUTICAL,目录号:RH1704)
  7. 聚维酮碘溶液(YING YANAN CHEMICAL PHARMACEUTICAL,目录号:S-166)
  8. 对乙酰氨基酚溶液(CENTER实验室,目录号:19746)
  9. Luxol快速蓝色染色试剂盒(Abcam,目录号:ab150675)
  10. 苏木精和曙红染色试剂盒(Vector Laboratories,目录号:H-3502)
  11. 甲氧苄啶 - 磺胺甲恶唑预混抗菌溶液(YUNG SHIN PHARM,目录号:TRI-004)
  12. 甲氧苄啶 - 磺胺甲恶唑抗菌注射液(见配方)

设备

  1. NYU-MASCIS减压冲击器,带鳄鱼和软件
  2. 2.5毫米大鼠冲击器尖端
  3. Scalpel手柄#4(DIMEDA Instrumente,目录号:06.104.00)
  4. 加热垫
  5. Adson齿形镊子(DIMEDA Instrumente,目录号:10.180.12)
  6. ALM自动牵引器(DIMEDA Instrumente,目录号:18.620.07)
  7. MAYO HEGAR针架(DIMEDA Instrumente,目录号:24.180.16)
  8. Littauer骨切割机(Stoelting,目录号:52167-80P)
  9. 操作剪刀(Shinetech,目录号:ST-S114PK)
  10. CMA/150温度控制器(CMA Microdialysis,型号:CMA 150,目录号:600)
  11. 干式灭菌器(Braintree Scientific,型号:Germinator 500,目录号:GER 5287-120V)
  12. 手术显微镜(Carl Zeiss,型号:Zeiss Stativ S3)
  13. 台式麻醉系统(AM Bickford,目录号:61020)
  14. EVA软泡沫垫(李春云企业,型号:FM 600T)

软件

  1. MAS 7.0版本
  2. Microsoft Windows 98操作系统

程序

道德声明:在本方案中使用成年女性Sprague-Dawley(SD)大鼠(225-250g)。涉及动物的所有手续均经台北退伍军人综合医院动物委员会(许可证编号IACUC 2014-137和IACUC 2015-253)批准,并符合国家卫生研究院概述的实验动物护理和使用指南

  1. 接触伤口内部的所有仪器(齿形钳,尖端,夹具,牵开器,针架,手术刀手柄和剪刀)必须使用干式灭菌器进行灭菌。
  2. 设置个人电脑并启动碰撞程序。
  3. 将大鼠放入感应室。
  4. 打开气流(以异氟烷为1〜1.5L/min为5%),然后监测大鼠直至躺卧。
  5. 将麻醉大鼠从腔室移至麻醉系统面罩,将异氟烷水平设定为1.5-2%。
  6. 使用直肠探针的电气温度控制器,在加热垫上保持大鼠的核心体温在36-37°C。
  7. 剃刮胸部区域,并将聚维酮碘溶液涂抹在剃光区域。
  8. 使用手术刀对背侧胸廓进行纵向切口,并切开椎旁肌,露出椎骨T7-T12。
    注意:T2的棘突长于其他任何一个。触摸皮肤下T2的位置以确定手指操作的大致位置(图1A)。
  9. 使用牵开器轻轻地将椎旁肌肉拉出脊柱。用Zeiss手术显微镜(7.5倍放大)进行椎板切除术。使用骨刀和齿形镊子切割并除去T8,T9和部分T10的骨骼。这将暴露脊髓的背表面而不破坏硬脑膜。 (图1B)
    注意:为避免碰撞器尖端撞击椎骨,必须拆下骨头,以获得超过2.5 mm的宽度,因为冲击器尖端的直径为2.5 mm。
  10. 两个稳定钳用于固定椎骨T7和T12的后棘突,并在挫伤期间支撑椎骨柱。
  11. 将T8-T10脊髓中心的大鼠放置在尖端下方(图1C)。撞击器的地面鳄鱼放在肌肉上,以形成尖端和脊髓之间的电导。
    注意:使用齿形镊子稍微推动夹紧的椎骨的中间,以确保椎骨夹紧好并且稳定(图1D)。
  12. 将尖端置于0mm处,然后向下放下杆,使尖端接触脊髓的硬脑膜(图1E,左侧)。当尖端触及硬膜时,撞击器的黑盒子会产生光线并发出嗡嗡声(图1E,右)。


    图1.椎板切除术,影响部位和动物护理。 :一种。在背侧胸廓表面显示的2 nd 胸椎(T2,黑圈)和9 胸椎(白色圆圈)的近似位置。 B.挫伤前脊髓已被T8-T10椎板切除术暴露(白色圆圈为脊椎部位)。 C.将大鼠放置在杆的中心,并固定夹具以显示椎骨T7和T12的棘突。 D.齿镊稍微推动夹紧椎骨的中间,以确保椎骨夹紧好且稳定。 E.通过插入的针将尖端置于0mm处;尖端可以升高并保持在特定高度(6.25,12.5,25或50mm;左)。当尖端触及硬脑膜时,撞击器的黑盒子会产生光线和嗡嗡声(右)。 F.脊髓部位出现硬膜下出血后T9椎体位置后挫伤。 G.肌肉用铬肠道连续关闭(左),并用尼龙缝线中断缝合皮肤(右)。 H.用一只手握住大鼠,用拇指轻轻挤压下腹部的膀胱,另一只手的前两根手指。

  13. 抬起杆,并用特定高度(包括6.25,12.5,25或50 mm)的插入针固定尖端。手动拉动插入的针脚,使杆在T9处由重力坠落到暴露的硬膜上,产生挫伤伤害。
    注意:软件可以记录的冲击器的参数为:冲击速度(Vi),绳索压缩距离(Cd),时间(Ct)和速率(Cr = Cd/Ct)。例如,应该以0.98m/sec的速度实现50mm高的冲击速度。通过软件显示,压缩距离应大约为2-3mm,如果大于3 mm,则说明脊髓未夹紧。 Ct呈现杆将脊髓压缩到最深点所需的时间。
  14. 挫伤后,硬膜下出血可以在硬脑膜下清楚看到(图1F)
  15. 抬起尖端,轻轻地从脊柱上释放夹具。
  16. 伤口连续关闭,肌肉用铬酸肠(4/0),并用尼龙缝线(3/0)缝合皮肤(图1G)。
  17. 关闭麻醉系统,然后将鼠标放回加热垫上的笼子上。
    注意:整个过程大约需要1小时,大鼠在去除异氟烷后的10-20分钟内应该被唤醒。稍微按下大鼠的爪子,SCI后24小时不应该出现后肢的反射作用。根据我们的经验,大鼠的死亡率低于5%。
  18. 监测大鼠,并通过日常观察提供术后护理,包括体重减轻,脱水和膀胱功能障碍。皮下注射3 ml无菌0.9%生理盐水进行再水化。术后5天皮下注射预防性抗生素(甲氧苄啶 - 磺胺甲恶唑抗菌注射液,0.9%生理盐水稀释1:30,2 ml/kg)。如果大鼠显示自残的迹象,则口服给药镇痛对乙酰氨基酚(65 mg/kg)。此外,如果大鼠显示严重的脚部损伤,我们通常会丢弃大鼠。
  19. 大鼠在常规动物房屋中照料,并保持在12小时的明暗周期。通过挤压下腹部,每天两次手动排空大鼠的膀胱(图1H) 注意:大多数大鼠在挫伤后1-3天显示血尿。如果血尿不能很好地清除,或在SCI后膀胱留下大量残留的尿液,可能会导致膀胱炎,感染甚至死亡。

数据分析

  1. 病变体积
    SCI导致气蚀和脱髓鞘,扩大了损伤程度(Poon等人,2007)。 Luxol fast blue(LFB)和苏木精和曙红(H& E)染色分别用于鉴别腔和有髓白质。染色程序遵循制造商的方案。用显微镜照相机以2.5倍放大的方式将图像从头端到整个损伤部位的尾端照相。耻辱(50毫米高度)造成大部分灰质损失和少量白质保留(图2)

    图2. SCI后6周的组织学染色 其中一个代表性结果显示,SCI大鼠LFB染色(上)和H&E染色的连续载玻片(底部)(-2mm,0mm和2mm;比例尺=250μm)。

  2. 运动员得分
    Basso,Beattie和Bresnahan(BBB)野外得分用于评估后肢的运动(Basso等,1996)。简单地说,将大鼠放置在垫子上(尺寸为100×100×40mm),并通过BBB测试从0(无可观察到的后肢运动)到21(正常后肢运动)点进行评分。 BBB评分(0-8)的低端特征为每个后肢关节运动,中间(9-14)和高(15-21)的特征在于体重支撑,前肢后肢协调步进,脚趾间隙,主要的爪子位置和尾部位置(有关BBB分数的详细描述,请参见Basso等人的参考文献,1996)。使用摄像机每周进行一次行为分析,由盲人检查者进行记录。来自考官的每个后肢的每周得分一起平均得出一分。可以使用Bonferroni事后检验的双向方差分析(ANOVA)来比较不同等级(或治疗)的组。根据Basso等人的研究(1996),运动员得分在6.25毫米组最高,50毫米组最低(请参考步骤13)。 6.25毫米组在挫伤后3周内表现出最大功能恢复至正常运动附近。 12.5毫米组从无或轻微的后肢关节运动迅速恢复,在3周内稳步前进。相比之下,25毫米组在挫伤后表现出缓慢的运动改善。 50mm组在3-7天内显示瘫痪,并且在随后的6周内没有体重支持恢复。此外,Mestre等人(2015)和我们以前的研究在呕吐后6周(SD)的50只SD大鼠组中产生了类似的结果(视频1)(Chiu ,2016)。

    Video 1. Locomotor recovery in 50 mm group. The rat showed paralysis within 1-7 days and no weight-support recovery in the following 6 weeks.

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player

食谱

  1. 甲氧苄啶 - 磺胺甲恶唑抗菌注射工作液
    甲氧苄啶 - 磺胺甲恶唑,1卷
    抗生素溶液稀释于0.9%生理盐水,30体积/体积

致谢

这项研究得到了中国科学院博士后研究员的支持,台湾科技部(MOST 104-2314-B-010-012-MY3)和台北退伍军人总医院(105V-E6-001-MY3) -1)。我们也感谢蔡蔡莹博士帮助台北退伍军人总医院神经再生实验室,提供实验空间和设施。

参考

  1. Basso,DM,Beattie,MS and Bresnahan,JC(1996)。  使用纽约大学体重下降装置与横断面进行脊髓挫伤后的分级组织学和运动结果。 139(2):244-256。
  2. (2016)。免疫调节剂诱饵受体3改善脊髓损伤后的运动功能恢复。 J Neuroinflammation 13( 1):154.
  3. Kwon,BK,Oxland,TR和Tetzlaff,W。(2002)。脊髓再生研究中使用的动物模型。 Spine 27(14):1504-1510。
  4. Mestre,H.,Ramirez,M.,Garcia,E.,Martiñón,S.,Cruz,Y.,Campos,MG and Ibarra,A.(2015)。  Lewis,Fischer 344和Sprague-dawley大鼠显示脂质过氧化,运动恢复和摩擦脊柱的差异脊髓损伤后的保留。前方Neurol 6:108.
  5. Metz,GA,Curt,A.,van de Meent,H.,Klusman,I.,Schwab,ME and Dietz,V.(2000)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/10674754"target ="_ blank">大鼠体重下降挫伤模型的验证:人类脊髓损伤的比较研究神经损伤 17(1):1-17。
  6. Mothe,AJ和Tator,CH(2012)。 Advances用于脊髓损伤的干细胞治疗。 J Clin Invest 122(11):3824-3834。
  7. Oyinbo,CA(2011)。创伤性继发性损伤机制脊髓损伤:这种多重级联的核子。神经生物学实验(战争) 71(2):281-299。
  8. Po,PC,Gupta,D.,Shoichet,MS和Tator,CH(2007)。  剪辑压缩模型对胸部脊髓损伤有用:组织学和功能相关。 32> 2853-9。Spine
  9. Singh,A.,Tetreault,L.,Kalsi-Ryan,S.,Nouri,A.and Fehlings,MG(2014)。< a class ="ke-insertfile"href ="http://www.ncbi .nlm.nih.gov/pubmed/25278785"target ="_ blank">创伤性脊髓损伤的全球流行率和发病率。临床流行病学6:309-331。
  10. Young,W.(2002)。  脊髓挫伤模型。 Prog Brain Res 137:231-255。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chiu, C., Cheng, H. and Hsieh, S. E. (2017). Contusion Spinal Cord Injury Rat Model. Bio-protocol 7(12): e2337. DOI: 10.21769/BioProtoc.2337.
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