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Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish
采用各种脊髓损伤模型研究成年斑马鱼中的细胞再生   

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Abstract

Spinal cord injury (SCI) in mammals leads to failure of both sensory and motor functions, due to lack of axonal regrowth below the level of injury as well as inability to replace lost neural cells and to stimulate neurogenesis. In contrast, fish and amphibians are capable of regenerating a variety of their organs like limb/fin, jaw, heart and various parts of the central nervous system (CNS). Zebrafish embryo and adult has become a very popular model to study developmental biology, cell biology and regeneration for various reasons. Adult zebrafish, one of the most important vertebrate models to study regeneration, can regenerate many of their body parts like fin, jaw, heart and CNS. In the present article we provide information on how to inflict different injury modalities in adult fish spinal cord. Presently, the significant focus of mammalian SCI is to use crush and contusion injury. To generate an entity comparable to the mammalian mode of injury, we have introduced the crush model in adult zebrafish along with complete transection injury, which is also known to be a valuable model to study axonal regeneration. Here we provide full description of the highly reproducible surgical procedures including some representative results. This protocol has been adapted from our previous publications, viz. Hui et al., 2010 and Hui et al., 2014. Briefly, we have described the two different injury modalities, crush and complete transection, and demonstrated the outcome of inflicting these injuries in the adult zebrafish cord by histological analysis of the tissues.

Keywords: Spinal cord(脊髓), Zebrafish(斑马鱼), Crush injury(挤压伤), Transection injury(横断损伤), Regeneration(再生)

Background

Any injury to mammalian spinal cord leads to the devastating consequence of paralysis and loss of function. In contrast to mammals, injury response in zebrafish cord is quite different, resulting in repair and regeneration of cord followed by functional recovery. A variety of lesioning protocols have been employed to study spinal cord injury and functional recovery in lower vertebrates (Holtzer 1956; Egar and Singer, 1972; Filoni et al., 1984; Becker et al., 1997; Margotta et al., 1991; Hui et al., 2010; Sîrbulescu and Zupanc, 2011) in last five decades. Among them the most popular experimental protocol to study spinal cord regeneration has been tail amputation (Holtzer, 1956; Egar and Singer, 1972; Filoni et al., 1984; Margotta et al., 1991; Sîrbulescu and Zupanc, 2011). Tail amputation involves complete removal of the caudal part of tail, where muscle, skin, bone and cartilages are also removed along with spinal cord. There is complete regeneration of tail along with the spinal cord leading to functional recovery after tail amputation and a substantial progress has been made to understand the cellular mechanism of spinal cord regeneration. But there are drawbacks of using this model. The major criticism against this model is that, it does not appropriately mimic SCI in human, since there is no tail structure in humans and the nature of the injury is different.

In teleosts, the most important lesion paradigm that has been widely used to study spinal cord regeneration is complete transection (Becker et al., 1997; Goldshmit et al., 2012). Transection refers to complete severing of cord which can often lead to spinal shock in humans. It occurs rarely in comparison to hemisection which commonly occurs in gunshot wounds. Transection could be the appropriate model to study axonal regeneration since there is no axonal sparing after transection injury and some believe that axonal sparing itself could augment regeneration in mammals (Basso et al., 1996).

On the other hand, compression and crush injuries are most prevalent in mammals under experimental conditions and in human accidental injury conditions (Thuret et al., 2006). In search for an appropriate injury model to study the regeneration in teleost, we successfully established a standardized crush injury model in zebrafish, which is comparable to the mammalian mode of injury (Hui et al., 2010). Among all the experimental paradigms mentioned, standardized crush injury is the most suitable model to understand both the mammalian and the teleostean scenario compared to transection or tail amputation models. The outcome of crush injury varies when compared to transection injury, as in crush injury secondary degenerative response elicits axonal degeneration, whereas in transection injury axonal tracts are disengaged almost immediately after injury.

Materials and Reagents

  1. Pyrex Glass Petri dishes (150 x 20 mm) (Corning, catalog number: 3160-152BO )
  2. Pyrex crystallizing dish (Corning, catalog number: 3140-150 )
  3. Wheaton Coplin staining jars (Sigma-Aldrich, catalog number: S6016 )
  4. Moist tissue paper
  5. Needle
  6. Single edged surgical blade (Sigma-Aldrich, catalog number: S2771 )
  7. Cotton swabs, sterile (6 inch) (Medline Industries, catalog number: MDS202000Z )
  8. Plastic Pasteur pipettes (BRAND, catalog number: 747755 )
  9. Phosphate buffered saline (PBS), pH 7.4 (Sigma-Aldrich, catalog number: P4417 )
  10. Tricaine (MS222) (Sigma-Aldrich, catalog number: E10521 )
  11. Zebrafish Aquarium System water
  12. 0.1% cresyl violet solution (Sigma-Aldrich, catalog number: C5042 )
  13. 0.1% luxol fast blue solution (Sigma-Aldrich, catalog number: L0294 )
  14. 4% paraformaldehyde (Sigma-Aldrich, catalog number: 158127 )
  15. 0.5 M EDTA (Sigma-Aldrich, catalog number: E9884 )
  16. Paraffin (Sigma-Aldrich, catalog number: 327212 )
  17. Xylene (Sigma-Aldrich, catalog number: 534056 )
  18. 0.05% lithium carbonate solution (Sigma-Aldrich, catalog number: 255823 )
  19. Graded ethanol (Sigma-Aldrich, catalog number: 24102 )
    Note: This product has been discontinued.
  20. Glacial acetic acid (Sigma-Aldrich, catalog number: A6283 )

Equipment

  1. Leica rotary microtome (Leica Biosystems Nussloch, model: RM 2125RTS )
  2. Student Dumont #5 forceps (Sigma-Aldrich, catalog number: F6521 )
  3. Micro dissectingspring scissors (Harvard Apparatus, catalog number: 728500 )
  4. Stereozoom dissecting microscope (Olympus, models: SZX7 and SZ51 )

Procedure

Note: The present study was carried out according to the guidelines provided by CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environments and Forests, Government of India). Surgery protocol was approved by the Institutional Animal Ethics Committee of our department under registration with CPCSEA (CPCSEA/ORG/CH/REG NO.925/295).

  1. Maintenance of fish
    Zebrafish stock population is either bred in our animal house facility or obtained from local pet shop. Fish are kept in separate groups of 15 in the aquatic system maintained at 28 °C on a 14 h light and 10 h dark cycle.
  2. Preparation of surgical plates
    106 mm glass Petri dish is used and moist tissue paper is placed in it, where the anesthetized fish is laid laterally (Figure 1a; Video 1). The gill is covered with tissue paper soaked in water or PBS.
  3. Fishes of same size and age (approximately 3-4 cm in length, 4-6 months old) are anaesthetized by dipping them in 0.02% tricaine (MS222) for 3-5 min (long exposure like 10 min or more could affect post-surgical recovery of spinal cord injured fish) at room temperature. It is important to check that fish are completely anaesthetized prior to surgery. After placing the anaesthetized fish on the Petri dish, the body is touched with a needle and absence of twitching movement confirms complete anaesthesia. Petri dish with anaesthetized fish is placed under stereozoom dissecting microscope and surgery is performed under the microscope.
  4. Surgery protocol
    1. Crush injury:
      1. A longitudinal incision is given with a single-edged blade on the second stripe of the fish body at the level of the dorsal fin, which corresponds to the 15/16th vertebrae (Figure 1b; Video 1).
      2. After making the cutaneous wound (Figure 1c; Video 1), one has to scrape muscle sidewise to reach the vertebral column, which resides at a much deeper level. Blood coming to the wound site can be cleared by using a sterile cotton swab (Figure1d; Video 1).


        Figure 1. Making a cutaneous wound before spinal cord injury. a. An anaesthetized adult zebrafish is placed laterally over a moist tissue paper before inflicting spinal cord injury; b. The pigmented stripped region (PR) below dorsal fin (DF) is the area where the cutaneous wound is made; c. Making a cutaneous wound by a sharp blade; d. Clearing the blood from wound area by using a sterile cotton swab; e. Tearing the muscles deep inside the wound using forceps to reach and clearly visualize the spinal cord.

        Video 1. Inflicting spinal cord injury in adult zebrafish

      3. Vertebral column is a hard but translucent tissue and spinal cord, encased within the vertebrae, is visible as the meninges are pigmented. After clearing the surrounding tissues, vertebral column is crushed dorso-ventrally with Dumont forceps #5 for 1 sec (Video 1; Figures 2 and 4, where the injury epicentre is marked with an asterisk).


        Figure 2. Crush injury to spinal cord using Dumont #5 forceps. a. Holding the forceps in stretch to clearly visualize the spinal cord before performing injury; b. Inflicting crush injury by holding the full spinal cord dorso-ventrally using Dumont #5 forceps for 1 sec; c. Covering the vertebral wound with muscle and skin; d. Higher magnification view of spinal cord inside the wound before injury; e. Higher magnification view of the spinal cord at the wound site during crush injury; f. Higher magnification view of the spinal cord inside the wound after crush injury. Star indicates the injury epicentre.

      4. One can use the bony projections of vertebral column and mark the corresponding fin rays of dorsal fin as a land mark. We cannot perform laminectomy in zebrafish spine, as we do while inflicting SCI in mouse, because the vertebral column is too thin here. After completing injury, surrounding muscle tissues are placed back to cover the injury site and only a single suture can be given through skin to reduce the wound size.
      5. Fishes are returned into shallow water in a Petri dish and water is gently blown over the gills using a plastic pipette so that fish can recover from anaesthesia quickly and can swim on their own. Fishes are finally transferred to larger tanks and allowed to regenerate for a specified period of time at 28 °C.
    2. Transection injury
      1. Similar to crush injury a longitudinal incision is given on the skin laterally with a single-edged blade on the second stripe of the fish body at the level of dorsal fin, which corresponds to the 15/16th vertebrae.
      2. After carefully scrapping the muscle beneath the wound, spinal cord encased in the vertebral column can be visualised clearly. On reaching the vertebral column deep to the wound site, the spinal cord is completely transected using a micro-dissecting spring scissors (Figures 3 and 4).
      3. As spinal cord is a soft spongy tissue, a sharp cut through the gap between the two vertebrae by the two blades of micro-scissors is enough to ensure complete transection separating the rostral and caudal stumps completely from each other. The injury epicentre is marked with an asterisk in Figure 4c. The injury would lead to paralysis of posterior part of the body. After completing injury procedures, surrounding muscle tissues are placed back to cover the vertebral wound. The suture may be given through the skin (Figure S1) or tissue glue can be used (optional) to reduce the wound size. Fishes are returned to a shallow water tank and water is blown gently over the gills using a plastic pipette so that fish can recover from anaesthesia quickly.
      4. Fishes are finally transferred to larger tanks and allowed to regenerate for a specified period of time at 28 °C.


        Figure 3. Transection injury to spinal cord using a micro-scissor. a. Holding the forceps in open position to clearly visualize the spinal cord before inflicting injury; b. Performing transection injury by completely severing the spinal cord into rostral and caudal stumps by using fine spring scissors; c. Covering the vertebral wound with muscle and skin; d. Higher magnification view of the spinal cord before injury; e. Higher magnification view of the spinal cord during transection injury; f. Higher magnification view of the spinal cord after transection injury. Star indicates the injury epicentre.


        Figure 4. Uninjured and injured spinal cord after crush and transection injury. a. Higher magnification picture of the uninjured spinal cord. b. Higher magnification picture of the spinal cord after crush injury. c. Higher magnification picture of the spinal cord after transection injury. Star indicates the injured region. SC = Spinal cord, VC = Vertebral column.

  5. Histological analysis to study time course after injury
    Luxol fast blue and cresyl violet staining (0.05% lithium carbonate solution and glacial acetic acid are used for differentiation step) is performed on the spinal cord tissue sections according to the standard Klüver-Barrera protocol (Sheehan and Hrapchak, 1980). Both uninjured and injured fishes are dissected, spinal cord taken out, fixed in 4% paraformaldehyde, decalcified in 0.5 M EDTA, passed through graded alcohol and xylene and embedded in paraffin (Hui et al., 2010). Time course analysis of stained section in uninjured cord shows relative distribution of white matter and grey matter as depicted in Figure 5a. Anatomical distribution of neurons in the subependymal and axons and cell bodies in white matter are obvious. Details of the cellular components are discussed in our previous publication (Hui et al., 2010). Here substantial tissue loss is shown at the early stages after crush injury (Figure 5b) with some tissue sparing, a characteristic feature of the crush injury model. Time course cellular response depends on the degree and extent of injury. In this case, as the transection injury is very clean, not much tissue has been lost and the stump tissues are very closely apposed (Figure 5c). Regeneration is very impressive and the time frame does not vary much when compared with crush injury. 
     

    Figure 5. Luxol fast blue staining in spinal cord tissue. Histological sections of uninjured spinal cord (a), spinal cord 3 days after crush injury (b) and spinal cord 3 days after transection injury (c) stained with luxol fast blue and cresyl violet. Please note that there are some spared tissues at the injury epicentre in b, whereas two separate stump tissues are clearly visible in c, with no axonal sparing. GM = Grey matter region of spinal cord, WM = white matter region of spinal cord, N = neuron, SEP = sub-ependyma, EP = ependyma. Star indicates the approximate injury epicentre. Scale bar=100 µm (a-c).

Data analysis

Injury protocols were standardized in our laboratory where hundreds of animals have been used for our array analysis (Hui et al., 2014). We could injure 30-40 animals in a day. All the histological analysis with the spinal cord tissues were repeated 3-5 times. The pictures shown here are not repeated in other publications. However, time course analysis of regeneration in zebrafish cord was previously published (Hui et al., 2010, http://www.suklaghosh.com).

Notes

  1. Two very simple yet standardized protocols for inflicting crush and transection injury in adult zebrafish cord have been described. The reproducibility of these injury modalities can be followed by analysing the tissue sections stained with different staining protocols like luxol fast blue and cresyl violet or Mallory’s trichrome staining or even routine hematoxylin/eosin staining.
  2. Staining, particularly using longitudinal section of the spinal cord tissue, enable us to characterize the nature of injury and visualize cellular response(s) at the injury epicentre and the adjoining area after injury and compare with the normal part of the same cord.

Acknowledgments

Both these injury protocols described here are generated at Sukla Ghosh’s laboratory, Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India. Grant support received for the work was from DBT (Govt. of India, BT/PR5489/AAQ/03/245/2004). Dr. Subhra Prakash Hui was recipient of Senior Research Fellowship from CSIR (Govt. of India). The spinal cord crush injury protocol in zebrafish has been adapted from our previous publications (Hui et al., 2010; 2014).

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. Becker, T., Wullimann, M. F., Becker, C. G., Bernhardt, R. R. and Schachner, M. (1997). Axonal regrowth after spinal cord transection in adult zebrafish. J Comp Neurol 377(4): 577-595.
  3. Egar, M. and Singer, M. (1972). The role of ependyma in spinal cord regeneration in the urodele, Triturus. Exp Neurol 37(2): 422-430.
  4. Filoni, S., Bosco, L. and Cioni, C. (1984). Reconstitution of the spinal cord after ablation in larval Xenopuslaevis. Acta Embryol Morphol Exp 5(2): 109-129.
  5. Goldshmit, Y., Sztal, T. E., Jusuf, P. R., Hall, T. E., Nguyen-Chi, M. and Currie, P. D. (2012). Fgf-dependent glial cell bridges facilitate spinal cord regeneration in zebrafish. J Neurosci 32(22): 7477-7492.
  6. Holtzer, S.W. (1956). The inductive activity of the spinal cord in urodele tail regeneration. J Morphol 99: 1-39.
  7. Hui, S. P., Dutta, A. and Ghosh, S. (2010). Cellular response after crush injury in adult zebrafish spinal cord. Dev Dyn 239(11): 2962-2979.
  8. Hui, S. P., Sengupta, D., Lee, S. G., Sen, T., Kundu, S., Mathavan, S. and Ghosh, S. (2014). Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish. PLoS One 9(1): e84212.
  9. Margotta, V., Fonti, R., Palladini, G., Filoni, S. and Lauro, G. M. (1991). Transient expression of glial-fibrillary acidic protein (GFAP) in the ependyma of the regenerating spinal cord in adult newts. J Hirnforsch 32(4): 485-490.
  10. Sheehan, D. C. and Hrapchak, B. B. (1980).Theory and practice of histotechnology. Battelle.
  11. Sîrbulescu, R. F. and Zupanc, G. K. (2011). Spinal cord repair in regeneration-competent vertebrates: adult teleost fish as a model system. Brain Res Rev 67(1-2): 73-93.
  12. Thuret, S., Moon, L. D. and Gage, F. H. (2006). Therapeutic interventions after spinal cord injury. Nat Rev Neurosci 7(8): 628-643.

简介

哺乳动物的脊髓损伤(SCI)导致感觉和运动功能的失败,这是由于缺乏低于损伤水平的轴突再生以及不能代替失去的神经细胞和刺激神经发生。相比之下,鱼和两栖动物能够再生各种器官,如肢体/鳍,下巴,心脏和中枢神经系统(CNS)的各个部分。斑马鱼胚胎和成人已经成为一个非常受欢迎的模型,研究发育生物学,细胞生物学和再生由于各种原因。成年斑马鱼是研究再生的最重要的脊椎动物模型之一,可以再生许多身体部位,如鳍,下巴,心脏和CNS。在本文中,我们提供如何在成年鱼脊髓造成不同的损伤方式的信息。目前,哺乳动物SCI的重点是使用挤压和挫伤损伤。为了产生与哺乳动物损伤模式相当的实体,我们已经在成年斑马鱼中引入了粉碎模型以及完全横断损伤,其也被认为是研究轴突再生的有价值的模型。在这里,我们提供高度可重复的手术程序的完整描述,包括一些有代表性的结果。此协议已经从我们以前的出版物, viz 改编。简而言之,我们描述了两种不同的损伤模式,挤压和完全横切,并证实了造成的结果
关键字:脊髓,斑马鱼,挤压伤,横切损伤,再生

[背景] 对哺乳动物脊髓的任何损伤导致瘫痪和功能丧失的毁灭性后果。与哺乳动物,斑马鱼绳的损伤反应是相当不同,导致线的修复和再生,然后功能恢复。已经使用多种损伤方案来研究脊髓损伤和下脊椎动物中的功能恢复(Holtzer 1956; Egar和Singer,1972; Filoni等人,1984; Becker等人,在过去的五十年中,人们已经开发出了一种新的方法,该方法可以用于治疗和预防骨质疏松症。其中最常见的用于研究脊髓再生的实验方案已经是尾截肢术(Holtzer,1956; Egar和Singer,1972; Filoni等人,1984; Margotta等人 >,1991;Sîrbulescuand Zupanc,2011)。尾截肢包括完全去除尾部的尾部,其中肌肉,皮肤,骨和软骨也随着脊髓一起被去除。尾部截肢后,尾部与脊髓完全再生,导致功能恢复,并且已经进行了实质性的进展以了解脊髓再生的细胞机制。但是有使用这个模型的缺点。对这种模型的主要批评是,它不适当地模仿人类的SCI,因为人类没有尾部结构,受伤的性质是不同的。在硬骨鱼中,已经广泛用于研究脊髓再生的最重要的病变范例是完全横断(Becker等人,1997; Goldshmit等人, 2012)。横断是指完全切断绳索,这通常会导致人类脊髓休克。与通常发生在枪伤中的半切断相比,它很少发生。横断可以是研究轴突再生的合适模型,因为在横切损伤后没有轴突保留,并且一些人认为轴突保留本身可以增加哺乳动物的再生(Basso等人,1996)。
 另一方面,压迫和挤压损伤在实验条件下和人类意外损伤条件下在哺乳动物中最普遍(Thuret等人,2006)。在寻找适当的损伤模型以研究硬骨鱼中的再生,我们成功地在斑马鱼中建立了标准化的挤压损伤模型,其与哺乳动物损伤模型相当(Hui等人,2010)。在所有提到的实验范例中,标准化挤压损伤是最理想的模型,以了解哺乳动物和teleostean情况相比,横断或尾截肢模型。挤压损伤的结果与横断损伤相比不同,因为挤压损伤继发性退化反应引起轴突变性,而在横切损伤中,轴突束在损伤后几乎立即脱离。

关键字:脊髓, 斑马鱼, 挤压伤, 横断损伤, 再生

材料和试剂

  1. Pyrex玻璃培养皿(150×20mm)(Corning,目录号:3160-152BO)
  2. Pyrex结晶皿(Corning,目录号:3140-150)
  3. Wheaton Coplin染色瓶(Sigma-Aldrich,目录号:S6016)
  4. 湿巾纸

  5. 单刃手术刀片(Sigma-Aldrich,目录号:S2771)
  6. 棉签,无菌(6英寸)(Medline Industries,目录号:MDS202000Z)
  7. 塑料巴斯德移液管(BRAND,目录号:747755)
  8. 磷酸盐缓冲盐水(PBS),pH 7.4(Sigma-Aldrich,目录号:P4417)
  9. Tricaine(MS222)(Sigma-Aldrich,目录号:E10521)
  10. 斑马鱼水族馆系统水
  11. 0.1%甲酚紫溶液(Sigma-Aldrich,目录号:C5042)
  12. 0.1%luxolfast blue溶液(Sigma-Aldrich,目录号:L0294)
  13. 4%多聚甲醛(Sigma-Aldrich,目录号:158127)
  14. 0.5M EDTA(Sigma-Aldrich,目录号:E9884)
  15. 石蜡(Sigma-Aldrich,目录号:327212)
  16. 二甲苯(Sigma-Aldrich,目录号:534056)
  17. 0.05%碳酸锂溶液(Sigma-Aldrich,目录号:255823)
  18. 分级乙醇(Sigma-Aldrich,目录号:24102) 注意:此产品已停产。
  19. 冰乙酸(Sigma-Aldrich,目录号:A6283)

设备

  1. 徕卡旋转切片机(Leica Biosystems Nussloch,型号:RM 2125RTS)
  2. Student Dumont#5镊子(Sigma-Aldrich,目录号:F6521)
  3. 微解剖剪刀(Harvard Apparatus,目录号:728500)
  4. 立体倍率解剖显微镜(Olympus,型号:SZX7和SZ51)

程序

注意:本研究是根据CPCSEA(动物实验控制和监督委员会,印度政府环境和森林部)提供的指南进行的。手术方案由我们部门的机构动物伦理委员会批准,注册于CPCSEA(CPCSEA/ORG/CH/REG NO.925/295)。

  1. 鱼的保养
    斑马鱼种群是在我们的动物房设施繁殖或从当地宠物商店获得。在保持在28℃的水生系统中,鱼在14小时光照和10小时黑暗循环下分成15组保持。
  2. 手术板的准备
    使用106mm玻璃培养皿,并且将潮湿的薄纸放置在其中,其中麻醉的鱼横向放置(图1a;视频1)。鳃由用水或PBS浸泡的薄纸覆盖
  3. 将相同大小和年龄(长度约3-4cm,4-6个月大)的鱼通过将其浸入0.02%三卡因(MS222)3-5分钟(长时间暴露如10分钟或更长可能影响后 - 外科手术恢复脊髓损伤的鱼)。重要的是检查鱼在手术前是否完全麻醉。将麻醉的鱼置于培养皿上后,用针触摸身体,没有抽搐运动证实完全麻醉。将麻醉鱼的培养皿置于立体倍率解剖显微镜下,并在显微镜下进行手术。
  4. 手术协议
    1. 挤压伤:
      1. 在背鳍的水平处,在鱼体的第二条上用单刃刀片进行纵向切口,其对应于15/16脊椎(图1b;视频1) 。
      2. 在进行皮肤伤口(图1c;视频1)之后,必须侧向地刮擦肌肉以到达位于更深的水平的脊柱。可以使用无菌棉签清除进入伤口部位的血液(图1d;视频1)

        图1.脊髓损伤前皮肤创伤。将麻醉的成年斑马鱼侧面放置在潮湿的组织纸上,然后施加脊髓损伤; b。背鳍(DF)下面的着色剥离区域(PR)是制造皮肤伤口的区域; C。用锋利的刀片做皮肤伤口; d。使用无菌棉签清除伤口区域的血液; e。使用镊子撕开伤口深处的肌肉,以达到并清楚地看到脊髓
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      3. 脊柱是硬的但是半透明的组织,并且被包在椎骨内的脊髓随着脑膜着色而可见。清除周围组织后,用Dumont镊子#5将椎柱压碎1秒(视频1;图2和图4,其中损伤中心标有星号)。


        图2.使用Dumont#5镊子挤压脊髓损伤。保持镊子拉伸清楚地可视化脊髓之前执行损伤; b。通过使用Dumont#5镊子从背侧保持全脊髓持续1秒造成挤压伤; C。用肌肉和皮肤覆盖脊椎伤口; d。损伤前伤口内脊髓的放大倍数视图; e。挤压损伤期间伤口部位的脊髓的更高放大视图; F。在伤口损伤后的伤口内的脊髓的更高放大视图。星号表示受伤中心。

      4. 可以使用脊柱的骨突并且将背鳍的相应鳍射线标记为标记。我们不能在斑马鱼脊椎中进行椎板切除术,因为我们在小鼠中造成SCI时,因为脊柱在这里太薄了。在完成损伤后,将周围的肌肉组织放回到覆盖损伤部位,并且可以通过皮肤给予单个缝合线以减小伤口尺寸。
      5. 将鱼放回培养皿中的浅水中,并使用塑料移液管将水轻轻地吹到鳃上,使得鱼可以快速从麻醉中恢复并且可以自己游泳。鱼最终转移到更大的罐中,并允许在28℃下再生指定的时间段。
    2. 横断损伤
      1. 类似于挤压伤,在位于背鳍水平处的鱼体的第二条上用单刃刀片在皮肤上横向给予纵向切口,其对应于15/16支椎骨。
      2. 在仔细刮除伤口下方的肌肉后,包在脊柱中的脊髓可以清楚地可视化。在到达伤口部位深处的脊柱时,使用微解剖弹簧剪刀完全横断脊髓(图3和图4)。
      3. 由于脊髓是软的海绵组织,通过微型剪刀的两个刀片穿过两个椎骨之间的间隙的锋利切口足以确保完全横断使得嘴侧和尾侧残端完全彼此分离。在图4c中用星号标记损伤中心。损伤会导致身体后部瘫痪。在完成损伤程序后,将周围的肌肉组织放回以覆盖脊椎伤口。缝线可以通过皮肤给予(图S1 )或组织胶(可选)以减少伤口尺寸。鱼返回浅水池,用塑料移液管轻轻地将水吹到鳃上,以便鱼能快速从麻醉中恢复。
      4. 鱼最终转移到更大的罐中,并允许在28℃下再生指定的时间段

        图3.使用微型剪刀对脊髓的横切损伤。将镊子保持在打开位置以在造成伤害之前清楚地观察脊髓; b。通过使用细弹簧剪刀将脊髓完全切断成喙和尾骨,执行横切损伤; C。用肌肉和皮肤覆盖脊椎伤口; d。损伤前脊髓的放大倍率视图; e。横断损伤时脊髓的放大视图; F。横断损伤后脊髓的放大视图。星号表示受伤中心。


        图4.挤压和横切损伤后未受伤和受伤的脊髓。未受伤的脊髓的放大图片。 b。挤压伤后脊髓的放大图片。 C。横断面损伤后脊髓的放大图像。星号表示受伤区域。 SC =脊髓,VC =脊柱
  5. 损伤后研究时程的组织学分析
    根据标准Klüver-Barrera方案(Sheehan和Hrapchak,1980)对脊髓组织切片进行Luxol快速蓝色和甲酚紫染色(0.05%碳酸锂溶液和冰醋酸用于分化步骤)。将未损伤的和受伤的鱼都解剖,取出脊髓,在4%多聚甲醛中固定,在0.5M EDTA中脱钙,通过梯度酒精和二甲苯并包埋在石蜡中(Hui等人,2010) 。未受伤的线中染色切片的时间过程分析显示白质和灰质的相对分布,如图5a所示。在白质中的亚依赖性和轴突和细胞体中的神经元的解剖分布是显而易见的。细胞组分的细节在我们先前的出版物(Hui等人,2010)中讨论。在这里,显示在挤压损伤后的早期阶段(图5b)的实质性组织损失与一些组织保留,挤压损伤模型的特征。时程细胞反应取决于损伤的程度和程度。在这种情况下,由于横断损伤非常干净,没有多少组织丢失,而残端组织非常接近(图5c)。再生是非常令人印象深刻的,与挤压伤相比,时间框架没有多少变化。 
     

    图5.脊髓组织中的Luxol快速蓝染色。在损伤后3天(b)和脊髓3天后的未损伤脊髓(a),脊髓的组织切片(c)用luxol快速蓝和甲酚紫染色。请注意,在b损伤震中有一些备用组织,而两个单独的树桩组织在c中清晰可见,没有轴突保留。 GM =脊髓灰质区,WM =脊髓白质区,N =神经元,SEP =亚室管膜,EP =室管膜。星形表示大致的损伤中心。比例尺=100μm(a-c)。

数据分析

损伤方案在我们的实验室中被标准化,其中数百个动物已经用于我们的阵列分析(Hui等人,2014)。我们可以在一天内伤害30-40只动物。所有的组织学分析与脊髓组织重复3-5次。这里显示的图片在其他出版物中不再重复。然而,斑马鱼中再生的时间过程分析先前已经公开(Hui等人,2010, http //www.suklaghosh.com )。

笔记

  1. 已经描述了用于在成年斑马鱼中造成挤压和横切损伤的两种非常简单但标准化的方案。这些损伤模式的再现性可以通过分析用不同染色方案(如luxol fast blue和甲酚紫或马洛里三色染色或甚至常规的苏木精/曙红染色)染色的组织切片来进行。
  2. 染色,特别是使用脊髓组织的纵向切片使我们能够表征损伤的性质,并且显示损伤后的损伤中心和邻接区域的细胞反应,并与同一条线的正常部分进行比较。 />

致谢

这里描述的这些损伤方案是在Sukla Ghosh的实验室,Department of Biophysics,Molecular Biology and Bioinformatics,University of Calcutta,Kolkata,India中产生的。从工作收到的资助支持来自DBT(印度政府,BT/PR5489/AAQ/03/245/2004)。 Dr. Subhra Prakash Hui从CSIR(印度政府)获得高级研究奖学金。斑马鱼中的脊髓压伤损伤方案已经从我们以前的出版物改编(Hui等人,2010; 2014)。

参考文献

  1. Basso,DM,Beattie,MS和Bresnahan,JC(1996)。  使用NYU体重降低装置与横切的脊髓挫伤后分级的组织学和运动结果。 Exp Neurol 139(2):244-256。
  2. Becker,T.,Wullimann,MF,Becker,CG,Bernhardt,RR和Schachner,M。(1997)。  在成年斑马鱼中脊髓横切后的轴突再生。 377(4):577-595。
  3. Egar,M。和Singer,M。(1972)。  37 37(2):422-430。
  4. Filoni,S.,Bosco,L。和Cioni,C。(1984)。  尿道中脊髓的诱导活动尾再生。 99:1-39
  5. Hui,SP,Dutta,A.和Ghosh,S。(2010)。  成年斑马鱼脊髓压伤后的细胞反应。 239(11):2962-2979。
  6. 图1显示了一个基于插入文件的插入文件,该插入文件包含了一个文件, http://www.ncbi.nlm.nih.gov/pubmed/24465396"target ="_ blank">在脊髓再生期间的基因组宽表达谱分析在斑马鱼中鉴定全面的细胞反应。 em> 9(1):e84212。
  7. Margotta,V.,Fonti,R.,Palladini,G.,Filoni,S.and Lauro,GM(1991)。  组织技术的理论与实践 Battelle 。
  8. Sîrbulescu,RF和Zupanc,GK(2011)。  脊柱在具有再生能力的脊椎动物中的脊髓修复:成体硬骨鱼作为模型系统。 Brain Res Rev 67(1-2):73-93。
  9. Thuret,S.,Moon,LD和Gage,FH(2006)。  脊髓损伤后的治疗性干预。 Nat Rev Neurosci 7(8):628-643。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Hui, S. P. and Ghosh, S. (2016). Various Modes of Spinal Cord Injury to Study Regeneration in Adult Zebrafish. Bio-protocol 6(23): e2043. DOI: 10.21769/BioProtoc.2043.
  2. Hui, S. P., Sengupta, D., Lee, S. G., Sen, T., Kundu, S., Mathavan, S. and Ghosh, S. (2014). Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish. PLoS One 9(1): e84212.
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