Photothrombosis引起的局部脑缺血脊髓损伤的小鼠模型
Summary
Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.
Abstract
Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.
Introduction
外伤性脊髓损伤（SCI）是影响SC的感觉和植物神经功能毁灭性的临床情况。患者存活SCI往往留下衰弱截瘫的显著影响他们的日常活动和生活1的质量。实验SCI车型已经在科学考察中不可或缺的工具来了解脊髓损伤的病理生理学和相关的神经修复过程。这些模型也被用于测试的目的是在功能恢复各种实验神经保护干预的临床前功效。目前，大部分车型SCI在实践应用中使用的物理钝力机械破坏和伤害的SC。这些方法包括SC 2挫伤，压缩，错位和横断。有人建议，该一次侧机械损伤的缺血集形式的继发性损伤在受伤的SC后 3,4。继发缺血的病因包括广泛组织变性，脑实质出血，有时被阻塞的血管通过组织水肿5-7。作为继发性损伤的SC的完整性被进一步影响的结果，神经元和胶质细胞严重受损功能和活力和凋亡导致在损伤的慢性阶段对梗死增长，类似于缺血半影中风后的生长8,9。几种机制一样兴奋性中毒，自由基的产生，和炎症已经报道负责缺血性细胞死亡以下脊髓损伤10,11。此外，SC缺血是胸腹主动脉瘤修复手术，往往导致截瘫的病人12,13的严重并发症。尽管有如此高的临床影响，目前可脊髓缺血具有高重复性非常少的机型。
核苷酸“> Photothrombosis（PT）是一种常用的方法，在大脑中14-20局灶性缺血的诱导。该技术是相当的非侵入性的，高度可再现的，并产生一个精确的局灶性缺血病灶在脑17的露出面积-21。这是通过光敏染料像玫瑰红（RB）16-20,22或赤藓红的B全身给药达到23接着血管适当光源局部照射。染料的光敏化引起的自由基的生成而扰乱平滑血管内皮的完整性，并引起血小板聚集，随后形成血栓。血流的阻塞由血栓导致在由容器24供给的区域的梗塞。由于减轻控制对强度和辐照的持续时间这一步骤产生高度均匀和可再现的梗塞。此外，这种方法可用于诱导梗死T对于不同的解剖位置，使缺血的作用空间（ 例如 ，灰质与白质）的理解。目前研究的目的是开发SC缺血小鼠容易和高度可再现的模式。我们描述SC缺血的小鼠模型的PT的过程。结果，从组织学和免疫证明，PT能有效诱导SC梗死，神经元缺失和胶质增生反应。
Protocol
Representative Results
Discussion
在这项研究中，我们描述了SC缺血的光化学模型。由于进展遗传工程一直存在于市售的转基因小鼠的激增已经使得有可能研究涉及在SC缺血病理生理学特异性基因的影响。这项研究的目的是开发脊髓缺血可重现小鼠模型。在这里，我们适应皮质PT型号诱导脊髓损伤的小鼠。手术后脊柱静脉和毛细血管对小鼠的背侧处的T11胸椎水平被暴露。然后RB，市售的光敏染料，通过眶后窦路线被注入…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作是由美国国立卫生研究院[格兰特没有支持。 R01NS069726]和美国心脏协会的资助以资助格兰特[格兰特没有。 13GRNT17020004]到SD。
Materials
| Rose Bengal | Sigma-Aldrich | 330000 | 20 mg/ml in sterile saline |
| C57Bl6/J | Jackson lab | 664 | 22-25g |
| Ketamine | VEDCO | NDC-50989-996-06 | 100 mg/ml |
| Xylazine | VEDCO | NDC-50989-234-11 | 100 mg/ml |
| Betadine solution | Purdue | NDC-67618-150-01 | 10% povidone iodine topical solution |
| Normal saline | Abott Laboratories | 04930-04-10 | For diluting RB, anaesthesia and for preventing tissue from drying |
| Artificial tears ointment | Rugby | NDC-0536-6550-91 | 83% white petrolatum |
| Ethanol | Decon labs.Inc | 2716 | 70% ethanol for disinfection |
| Metal halide lamp | EXFO, Canada | X-Cite 120 PC | Set power at 12% |
| Spring scissors | Fine Science Tool | 15000-10 | for minor dissection |
| Scissors (angled to side) | Fine Science Tool | 14063-011 | No. 3 handle |
| Standard scalpel | Fine Science Tool | 10003-12 | for removing muscle |
| Scalpel blade | Feather | 2976 | No. 10 |
| Forceps (curved) | Fine Science Tool | 11150-10 | for holding tissue |
| Forceps (straight) | Fine Science Tool | 11151-10 | for holding tissue |
| Needle holder | Fine Science Tool | 12002-12 | for suturing |
| Tissue adhesive glue | 3M Vetbond | 1469SB | to adhere to edges of the cut skin |
| Monofilament polypropylene | USSC Sutures | VP-521 | Size = 4-0 (for fascia) |
| Perma-hand silk | Ethicon | 683G | Size = 4-0 (for skin) |
| Micro drill | Roboz Surgical Instrument Co. Inc. | RS-6300 | with bone polishing drill bit |
| Laser doppler flowmeter | Moor Instruments | moorVMS-LDF1 | for monitoring change in blood flow |
| Heating pad | Fine Science Tool | 21052-00 | to prevent hypothermia |
| Lab-Jack | Fisher scientific | 14-673-50 | 4×4 in plate to adjust the height of the animal |
| X-Y gliding stage | Amscope | GT100 | for positioning the animal under microscope |
Riferimenti
- Cadotte, D. W., Fehlings, M. G. Spinal cord injury: a systematic review of current treatment options. Clin Orthop Relat Res. 469 (3), 732-741 (2011).
- Cheriyan, T., et al. Spinal cord injury models: a review. Spinal Cord. 52 (8), 588-595 (2014).
- Young, W. Secondary injury mechanisms in acute spinal cord injury. J Emerg Med. 11, 13-22 (1993).
- Crowe, M. J., Bresnahan, J. C., Shuman, S. L., Masters, J. N., Beattie, M. S. Apoptosis and delayed degeneration after spinal cord injury in rats and monkeys. Nat Med. 3 (1), 73-76 (1997).
- Soubeyrand, M., et al. Effect of norepinephrine on spinal cord blood flow and parenchymal hemorrhage size in acute-phase experimental spinal cord injury. Eur Spine J. 23 (3), 658-665 (2014).
- Soubeyrand, M., et al. Real-time and spatial quantification using contrast-enhanced ultrasonography of spinal cord perfusion during experimental spinal cord injury. Spine (Phila Pa 1976). 37 (22), E1376-E1382 (2012).
- Mautes, A. E., Weinzierl, M. R., Donovan, F., Noble, L. J. Vascular events after spinal cord injury: contribution to secondary pathogenesis). Phys Ther. 80 (7), 673-687 (2000).
- Liu, X. Z., et al. Neuronal and glial apoptosis after traumatic spinal cord injury. J Neurosci. 17 (14), 5395-5406 (1997).
- Liu, L., et al. An experimental study of cell apoptosis and correlative gene expression after tractive spinal cord injury in rats. Zhonghua Wai Ke Za Zhi. 42 (23), 1434-1437 (2004).
- Hirose, K., et al. Activated protein C reduces the ischemia/reperfusion-induced spinal cord injury in rats by inhibiting neutrophil activation. Ann Surg. 232 (2), 272-280 (2000).
- Oyinbo, C. A. Secondary injury mechanisms in traumatic spinal cord injury: a nugget of this multiply cascade. Acta Neurobiol Exp (Wars). 71 (2), 281-299 (2011).
- Guerit, J. M., Dion, R. A. State-of-the-art of neuromonitoring for prevention of immediate and delayed paraplegia in thoracic and thoracoabdominal aorta surgery). Ann Thorac Surg. 74 (5), S1867-S1869 (2002).
- Schepens, M. A., Heijmen, R. H., Ranschaert, W., Sonker, U., Morshuis, W. J. Thoracoabdominal aortic aneurysm repair: results of conventional open surgery. Eur J Vasc Endovasc Surg. 37 (6), 640-645 (2009).
- Braeuninger, S., Kleinschnitz, C. Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems. Exp Transl Stroke Med. 1, 8 (2009).
- Carmichael, S. T. Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx. 2 (3), 396-409 (2005).
- Dietrich, W. D., Ginsberg, M. D., Busto, R., Watson, B. D. Photochemically induced cortical infarction in the rat. 1. Time course of hemodynamic consequences. J Cereb Blood Flow Metab. 6 (2), 184-194 (1986).
- Zhang, W., et al. Neuronal protective role of PBEF in a mouse model of cerebral ischemia. J Cereb Blood Flow Metab. 30 (12), 1962-1971 (2010).
- Li, H., Zhang, N., Sun, G., Ding, S. Inhibition of the group I mGluRs reduces acute brain damage and improves long-term histological outcomes after photothrombosis-induced ischaemia. ASN Neuro. 5 (3), 195-207 (2013).
- Li, H., et al. Histological, cellular and behavioral assessments of stroke outcomes after photothrombosis-induced ischemia in adult mice. BMC Neurosci. 15, 58 (2014).
- Wang, T., Cui, W., Xie, Y., Zhang, W., Ding, S. Controlling the Volume of the Focal Cerebral Ischemic Lesion through Photothrombosis. American Journal of Biomedical Sciences. 2 (1), 33-42 (2010).
- Schroeter, M., Jander, S., Stoll, G. Non-invasive induction of focal cerebral ischemia in mice by photothrombosis of cortical microvessels: characterization of inflammatory responses. J Neurosci Methods. 117 (1), 43-49 (2002).
- Boquillon, M., Boquillon, J. P., Bralet, J. Photochemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema. J Pharmacol Toxicol Methods. 27 (1), 1-6 (1992).
- Kim, G. W., Lewen, A., Copin, J., Watson, B. D., Chan, P. H. The cytosolic antioxidant, copper/zinc superoxide dismutase, attenuates blood-brain barrier disruption and oxidative cellular injury after photothrombotic cortical ischemia in mice. Neuroscienze. 105 (4), 1007-1018 (2001).
- Schmidt, A., et al. Photochemically induced ischemic stroke in rats. Exp Transl Stroke Med. 4 (1), 13 (2012).
- Lang-Lazdunski, L., et al. Spinal cord ischemia. Development of a model in the mouse. Stroke. 31 (1), 208-213 (2000).
- Wang, Z., et al. Development of a simplified spinal cord ischemia model in mice. J Neurosci Methods. 189 (2), 246-251 (2010).
- Labat-gest, V., Tomasi, S. Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies. J Vis Exp. (76), (2013).
- Lu, H., et al. Induction and imaging of photothrombotic stroke in conscious and freely moving rats. J Biomed Opt. 19 (9), 96013 (2014).
- Seto, A., et al. Induction of ischemic stroke in awake freely moving mice reveals that isoflurane anesthesia can mask the benefits of a neuroprotection therapy. Front Neuroenergetics. 6 (1), (2014).
- Bell, M. T., et al. Toll-like receptor 4-dependent microglial activation mediates spinal cord ischemia-reperfusion injury. Circulation. 128 (11 Suppl 1), S152-S156 (2013).
- Smith, P. D., et al. The evolution of chemokine release supports a bimodal mechanism of spinal cord ischemia and reperfusion injury. Circulation. 126 (11 Suppl 1), S110-S117 (2012).
- Jia, Z., et al. Oxidative stress in spinal cord injury and antioxidant-based intervention. Spinal Cord. 50 (4), 264-274 (2012).
