Pseudofracture：急性周围组织的创伤模型
Summary
Pseudofracture，重现的无菌肌肉骨骼损伤的小鼠模型，可以为后期的长期创伤后的免疫反应的评价。本文介绍的模式一步一步的程序执行，包括允许多发性创伤的研究实验模型组合的潜力。
Abstract
创伤后有一个早期的超反应性的炎症反应，可导致多器官功能障碍和创伤患者的死亡率高;这种反应往往伴随着延迟免疫抑制，增加感染的临床并发症， 还可以增加死亡率1-9。许多研究已开始在免疫系统的反应，以评估这些变化的创伤。 10-15
免疫学研究的大力支持，通过多种转基因和基因敲除小鼠体内建模;这些菌株在详细调查援助，以评估参与免疫反应的分子途径 16-21
在实验小鼠创伤建模的挑战是长期的调查，在小鼠骨折的内固定技术，可以复杂，不容易重现 。22-30
这pseudofracture模式，一个很容易复制的创伤模型，克服了这些困难，免疫模仿下肢骨折的环境，同时允许在动物和长期生存的自由运动，没有持续的，长期使用麻醉。其目的是重新长骨骨折的特点，受伤的肌肉和软组织暴露不破坏原生骨受损的骨骼和骨髓。
pseudofracture模型由两部分组成：一个双边的后肢肌肉挤压伤，其次是注射到这些受伤的肌肉骨解决方案，。准备收获从一个年龄和体重相匹配的同源捐助两个后肢长骨骨的解决方案。然后，这些骨头粉碎，重悬于磷酸盐缓冲生理盐水创建骨解决方案。
双侧股骨骨折是四肢创伤中常用的和行之有效的模式，并pseudofracture模型的发展过程中比较模型。各种可用的骨折模型当中，我们选择使用我们的pseudofracture比较软组织损伤的骨折闭合方法，因为我们想无菌但周围组织的比例严重创伤模型。 31
失血性休克是一种严重创伤的设置共同发现，和全球的低灌注创伤模型增加了一个非常相关的元素。32-36的pseudofracture模型可以很容易地与失血性休克模型为多发性创伤的严重程度高的模型相结合37。
Protocol
1。仪器及手术现场准备： 所有的实验程序进行使用无菌操作技术。实验区开始之前，必须彻底清洗和消毒。台式，应进行消毒，空气干燥，然后用70％的酒精抹允许。将一个蓝色的手术垫和无菌区，敷在试点工作区。 所有材料和工具，高压灭菌消毒后方可使用。收到无菌注射器和针头。研究者应适当garbed白大褂，口罩，无菌手术手套。 <p class="jove_conte…
Discussion
Pseudofracture，重现的无菌肌肉骨骼损伤的小鼠模型，可以评估创伤后的免疫反应。通过休闲的长骨骨折的特点，pseudofracture模型免疫模仿下肢骨折环境：受伤的肌肉和软组织暴露不破坏原生骨骨和骨髓受损 38,39双相免疫反应，可以看出以下。 pseudofracture创伤早期hyperinflammatory的响应，可以看出在6个小时的延迟免疫抑制的第二个组件描述为大约48小时的低谷到高峰组成。这种模式有助于克服?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
资金来源/编号分子生物学失血性休克GM053789
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Surgical blue pad | Fisher Scientific | 50-7105 | ||
| Sterile Field dressings | Fisher Scientific | NC9517505 | ||
| Circulating heating pad 18″x26″ | Harvard | py872-5272 | ||
| Hot bead instrument sterilizer | VWR | 11156-002 | ||
| Stainless steel tray 8″ x 11″ | VWR | 62687-049 | ||
| Plexiglass boards (10x15x0.5cm) | University of Pittsburgh Machine shop | |||
| Tape rolls 1″ | Corporate Express | MMM26001 | ||
| 50cc conical tube | can be purchased through any global vendor | |||
| Straight side wide mouth jars (used as cap for nose cone) | VWR | 159000-058 | ||
| Oster A5 clippers w. size 40 blade | VWR | 10749-020 | ||
| Surgical scissors (straight – 12cm) | Fine Science Tools | 14068-12 | ||
| Hemostats curved -18cm | Harvard | 81331718 | ||
| Forceps (0.8mm-tip, curved-10cm) | Fine Science Tools | 11050-10 | ||
| Gauze 4″x4″ | can be purchased through any global vendor | |||
| 1.5cc microfuge tube | can be purchased through any global vendor | |||
| Ice bucket | can be purchased through any global vendor | |||
| Mortar and Pestle | Fisher | 12-961AA | ||
| 1cc syringe w/ 25G needle | Fisher Scientific | 14-826-88 | ||
| 20G needle | can be purchased through any global vendor | |||
| 1mL pipetteman | can be purchased through any global vendor | |||
| 1mL pipette tips | can be purchased through any global vendor | |||
| Falcon polystyrene 8ml tubes | VWR | 60819-331 | ||
| Sterilization pouch 3″x8″ | VWR | 24008 | ||
| Sterilization pouch 5″x10″ | VWR | 24010 | ||
| MacConkey II Agar plate | BD Biosciences | 221172 | ||
| Ethyl Alcohol – 200 proof | Pharmaco-AAPER | [70%] | ||
| Pentobarbital Sodium (Nembutol Sodium Solution) | Ovation | 70mg/kg | ||
| Aerrane (Isoflurane) | Baxter | 99.9% | ||
| Triadine Povidone Iodine (Betadine) | Triad disposables | |||
| Phosphate Buffered Saline (PBS) | ||||
| Buprenorphine HCl | Bedford Laboratories | 0.1mg/kg |
Referencias
- DeCamp, M. M., Demling, R. H. Posttraumatic multisystem organ failure. JAMA. 260, 530-534 (1988).
- Deitch, E. A. Multiple organ failure. Pathophysiology and potential future therapy. Ann Surg. 216, 117-134 (1992).
- Carrico, C. J., Meakins, J. L., Marshall, J. C. Multiple organ failure syndrome. Arch Surg. 121, 196-208 (1986).
- Hauser, C. J., Joshi, P., Jones, Q. Suppression of natural killer cell activity in patients with fracture/soft tissue injury. Arch Surg. 132, 1326-1330 (1997).
- Faist, E., Baue, A. E., Dittmer, H. Multiple organ failure in polytrauma patients. J Trauma. 23, 775-787 (1983).
- Baker, C. C., Oppenheimer, L., Stephens, B. Epidemiology of trauma deaths. Am J Surg. 140, 144-150 (1980).
- Faist, E., Kupper, T. S., Bakeer, C. L. Depression of cellular immunity after major injury its association with posttraumatic complications and its reversal with immunomodulation. Arch Surg. 121, 1000-1005 (1986).
- Lenz, A., Franklin, G. A., Cheadle, W. G. Systemic inflammation after trauma. Injury. 38, 1336-1345 (2007).
- Flohe, S., Flohe, S. B., Schade, F. G. Immune response of severely injured patients–influence of surgical intervention and therapeutic impact. Lang Arch Surg. 392, 639-648 (2007).
- Ayala, A., Wang, P., Ba, Z. F. Differential alterations in plasma IL-6 and TNF levels after trauma and hemorrhage. Am J Physiol. 260, R167-R171 (1991).
- Kalicke, T., Schlegel, U., Printzen, G. Influence of a standardized closed soft tissue trauma on resistance to local infection. An experimental study in rats. J Ortho Res. 21, 373-378 (2003).
- Kobbe, P., Vodovotz, Y., Kaczorowski, D. J. The role of fracture associated soft tissue injury in the induction of sytemic inflammation and remote organ dysfunction after bilateral femur fracture. J Ortho Trauma. 22, 385-390 (2008).
- Kobbe, P., Vodovotz, Y., Kaczorwoski, D. J. Pattern of cytokine release and evolution of remote organ dysfunction after bilateral femur fracture. Shock. 30, 43-47 (2008).
- Flohe, S. B., Flohe, S., Schade, F. U. Deterioration of the immune system after trauma: signals and cellular mechanisms. Inn. Immun. 14, 333-344 (2008).
- Maier, B., LeFering, R., Lhenert, M. Early versus late onset of multiple organ failure is associated with differing patterns of plasma cytokine biomarker expression and outcome after severe trauma. Shock. 28, 668-674 (2007).
- Mestas, J., Hughes, C. W. Of mice not men: differences between mouse and human immunology. J. Immunol. 172, 2731-2738 (2004).
- Hoth, J. J., Wells, J. D., Brownlee, N. A. Toll like receptor 4-dependent responses to lung injury in a murine model of pulmonary contusion. Shock. 31, 376-381 (2009).
- Matsutani, T., Samy, A. n. a. n. t. h. a., Rue, T. S., W, L. Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions. Am J Physiol Cell Physiol. 288, 1109-1116 (2005).
- Matsutani, T., Samy, A. n. a. n. t. h. a., Kang, T. S., S-C, . Mouse genetic background influences severity of immune responses following trauma-hemorrhage. Cytokine. 30, 168-117 (2005).
- Tsukamoto, T., Pape, H. C. Animal models for trauma research: What are the options. Shock. 31, 3-10 (2008).
- DeMaria, E. J., Pellicane, J. V., Lee, R. B. Hemorrhagic shock in endotoxin resistant mice : Improved survival unrelated to deficient production of tumor necrosis factor. J Trauma. 35, 720-724 (1993).
- Jamsa, T., Jalovaara, P., Peng, Z. Comparison of three-point bending test and peripheral quantitative computed tomography analysis in the evaluation of the strength of mouse femur. Bone. 23, 155-161 (1998).
- Bounarens, F., Einhorn, T. A. Production of a standard closed fracture in laboratory animal bone. J Ortho Res. 2, 97-101 (1984).
- Holstein, J. H., Menger, M. D., Culemann, U. Development of a locking femur nail for mice. J Biomech. 40, 215-219 (2007).
- Holstein, J. H., Garcia, P., Histing, T. Advances in the establishment of defined mouse models for the study of fracture healing and bone regeneration. J Orthop Trauma. 23, S31-S38 (2009).
- Histing, T., Garcia, P., Matthys, R. An internal locking plate to study intramembranous bone healing in a mouse femur fracture model. J Orthop Research. 28, 397-402 .
- Manigrasso, M. B., O’Connor, J. P. Characterization of a closed femur fracture model in mice. J Ortho Trauma. 18, 687-695 (2004).
- Mark, H., Bergholm, J., Nilsson, A. An external fixation method and device to study fracture healing in rats. Acta Orthop Scand. 74, 476-487 (2003).
- Bhandari, M., Shanghnessy, S. A minimally invasive percutaneous technique of intramedullary nail insertion in an animal model of fracture healing. Arch Orthop Trauma Surg. 121, 591-593 (2001).
- Sonanis, S. V., Lampard, A. L., Kamat, N. A simple technique to remove a bent femoral intramedullary nail and broken interlocking screw. J Trauma. 63, 435-438 (2007).
- Kobbe, P., Kaczorwoski, D. J., Vodovotz, Y. Local exposure of bone components to injured soft tissue induces Toll-like receptor 4-dependent systemic inflammation with acute lung injury. Shock. 30, 686-691 (2008).
- Bumann, M., Henke, T., Gerngross, H. Influence of haemorrhagic shock on fracture healing. Lang Arch Surg. 388, 331-338 (2003).
- Santry, H. P., Alam, H. B. Fluid resuscitation: past, present, and the future. Shock. 33, 229-241 (2010).
- Hierholzer, C., Billiar, T. R. Molecular mechanisms in the early phase of hemorrhagic shock. Lang Arch Surg. 386, 302-308 (2001).
- Chaudry, I., Ayala, A., Ertel, W. Hemorrhage and resuscitation: immunological aspects. Am J Physiol. 259, 63-678 (1990).
- Kawasaki, T., Hubbard, W. J., Choudhry, M. A. Trauma-hemorrhage induces depressed splenic dendritic cell functions in mice. J Immunol. 177, 4514-4520 (2006).
- Kohut, L., Darwiche, S. S., Frank, A. M., Brumfield, J. M., Billiar, T. R. Fixed volume or Fixed Pressure: A Murine Model of Hemorrhgaic Shock. J Vis Exp. , (2010).
- Menzel, C. L., Pfeifer, R., Darwiche, S. S. Models of lower extremity damage in mice: time course of organ damage and immune response. J Surg Res. 166, e149-e156 (2011).
- Pfeifer, R., Kobbe, P., Darwiche, S. S. Role of hemorrhage in the induction of systemic inflammation and remote organ damage: Analysis of combined pseudo-fracture and hemorrhagic shock. J Orthop Res. 29, 270-274 (2011).
Tags
PseudofractureHyper-reactive Inflammatory ResponseMultiple Organ DysfunctionHigh MortalityDelayed ImmunosuppressionClinical ComplicationsInfectionMortalityImmunologic StudiesTransgenic MiceKnockout MiceIn Vivo ModelingMolecular PathwaysFracture Fixation TechniquesPseudofracture ModelExtremity Fracture EnvironmentDamaged Bone And Bone Marrow
Citar este artículo
Darwiche, S. S., Kobbe, P., Pfeifer, R., Kohut, L., Pape, H., Billiar, T. Pseudofracture: An Acute Peripheral Tissue Trauma Model. J. Vis. Exp. (50), e2074, doi:10.3791/2074 (2011).