参见作者原研究论文

本实验方案简略版
Jun 2018

本文章节


 

A Mouse Model of Postoperative Pain
小鼠术后疼痛模型   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Postoperative pain is highly debilitating and hinders recovery. Opioids are the main pain medication used for acute postoperative pain. Given the devastating opioid addiction and overdose epidemic across the US, non-opioid pain therapeutics are desperately needed. In order to develop novel, non-opioid therapies for the treatment of postoperative pain and identify the mechanisms underlying this pain, rodent models of incisional pain have been established. The protocol herein describes in detail how to create a mouse model of postoperative pain that was adapted from established protocols. This model of postoperative pain is frequently-used, highly reproducible, and results in peripheral and central nervous system alterations.

Keywords: Postoperative pain (术后疼痛), Inflammatory pain (炎症性疼痛), Hypersensitivity (超敏反应), Plantar incision (足底切口), Mouse (小鼠), Skin and muscle incision (皮肤和肌肉切口)

Background

Postoperative pain is a significant, worldwide problem. Approximately 234.2 million people undergo major surgeries each year (Weiser et al., 2008) and about 80% of patients experience acute postoperative pain (Gan, 2017). Of these, between 10% and 50% of patients, develop chronic pain that continues to severely impact their quality of life (Chapman and Vierck, 2017). One of the factors that are associated with the development chronic postoperative pain, but unlikely the cause, is the severity of acute pain experienced during the first postoperative week (Fletcher et al., 2015; Chapman and Vierck, 2017). Opioids are the main pain medication used for acute postoperative pain (Sen and Bathini, 2015; Tan et al., 2018). Given the opioid epidemic, non-opioid pain therapeutics are needed. Therefore, identifying the mechanisms that underlie acute postoperative pain is necessary for the development of optimal therapies for postoperative pain that may ultimately decrease the severity and/or incidence of chronic postoperative pain. Both rat (Brennan et al., 1996) and mouse (Pogatzki and Raja, 2003) models of acute incisional pain have been developed as preclinical models to identify the molecular, cellular and physiological mechanisms that underlie postoperative pain. However, a detailed description of the mouse model of postoperative pain is lacking. Here we describe in detail a mouse model of postoperative pain that requires incision of both the skin and muscle. Incision of both skin and muscle best mimics invasive surgery that causes intense acute pain and leads to chronic pain (Brennan, 2011; Chapman and Vierck, 2017). Furthermore, incision of skin and muscle (~6 days) creates hypersensitivity that lasts substantially longer than the skin-only (~3 days) incision model (Xu and Brennan, 2010). In this protocol, we provide detailed, step-by-step methods adapted from previous protocols (Brennan et al., 1996; Pogatzki and Raja, 2003) for development of a mouse model of postoperative pain.

Materials and Reagents

  1. Stainless steel sterile No. 11 surgical blade (World Precision Instruments, Feather Safety Razor Co. Ltd., catalog number: 504170)
  2. Sterile 5-0 nylon surgical sutures (AD Surgical, Unify, catalog number: S-N518R13)
  3. Surgical tape (3M, Transpore, catalog number: 1527-0)
  4. Cotton swab (VWR, Critical Swab, catalog number: 89031-270)
  5. Glad Press’n Seal (SAI Infusion Technologies, Glad, catalog number: PSS-70)
  6. Sterile nitrile gloves (Kimberly-Clark Professional, Kimtech Pure, catalog number: HC61170)
  7. Petri dishes [VWR, 14.5 and 9 663161, Greiner Bio-One, catalog numbers: 82050-912 (small) and 82050-600 (large)]
  8. Sterile gauze (Allied Medical, Ardes, catalog number: GA441221)
  9. Sharpie extra fine point permanent marker (Staples, Sharpie, catalog number: 37001) 
  10. 8-16 week old C57BL/6J mice (JAX, catalog number: 000664)
  11. Bacitracin zinc ointment (Fougera Pharmaceuticals Inc, catalog number: 0168-0011-04)
  12. Isoflurane (Clipper distributing company LLC., Phoenix, catalog number: 0010250)
  13. 75% ethanol (Fisher Scientific, Decon Laboratories, Inc., catalog number: 22-281-562)
  14. Surgical scrub 7.5% povidone-iodine (Betadine, Veterinary, catalog number: 67618-154-01)
  15. Eye lube (Patterson Veterinary, Optixcare Ophthalmic, catalog number: 07-893-2779)

Equipment

  1. 1,000 ml beaker (VWR, PYREX, catalog number: 13912-284)
  2. #55 Dumostar Forceps (Fine Science Tools, Dumont, catalog number: 11295-51)
  3. Scalpel handle (Fine Science Tools, catalog number: 10003-12)
  4. Iris Forceps, 10 cm, Curved, Serrated (World Precision Instruments, catalog number: 15915)
  5. Halsted Mosquito Hemostatic Forceps, 12.5 cm, Straight (World Precision Instruments, catalog number: 15920-G)
  6. Vannas Scissors, 8 cm, Curved (World Precision Instruments, catalog number: 14122)
  7. Small animal surgery board (Braintree Scientific, Inc., CD+, catalog number: ACD 014)
  8. Isoflurane dispenser (Highland Medical Equipment, Drager, catalog number: 16-7001)
  9. Sliding top isoflurane induction chamber (Kent Scientific Corporation, catalog number: VetFlo-0530LG)
  10. Heat Therapy Pump with Pad (Adroit Medical Systems, catalog number: HTP-1500)
  11. Isothermal pad (Braintree Scientific, Inc., Deltaphase, catalog number: DPIP)
  12. Digital calipers (VWR, catalog number: 62379-531)
  13. Steri 250 Bead Sterilizer Bead Bath (Lab Unlimited, Simon Keller Ltd., catalog number: 4AJ-6286283)
  14. Microwave (Emerson, 1,000 W, catalog number: B007Q45CIS)
  15. Home cage containing Aspen Sani Chips® (P.J. Murphy Forest and Products, Sani Chips®)

Software

  1. GraphPad Prism 7

Procedure

  1. Sterilize surgical area with ethanol and sterilize tools in bead bath of at least 325 °C for 30 s. Rest sterile tools, sutures, and blade on sterile gauze.
  2. Warm Heat Therapy Pump pad to 41 °C and warm the Isothermal pad in microwave for 1 min. Place a new, clean home cage on the Heat Therapy Pump pad. Place a laboratory paper towel over the warm Isothermal pad and cover with sterile Press’n Seal and surgery board.
  3. To induce anesthesia, put the mouse in the beaker chamber with paper towel on lid containing 1 ml of isoflurane (Figure 1) and wait for approximately 30 s or until the mouse can no longer right itself. Alternatively, an isoflurane induction chamber that is included with the isoflurane dispenser can be used to induce anesthesia.


    Figure 1. Brief inhalational anesthesia. The mouse was briefly anesthetized by inhalation in chamber with 1 ml isoflurane.

  4. Quickly remove the mouse from the isoflurane chamber and apply eye lube using a cotton swab to each eye. Further anesthetize the mouse with 1.5%-2% inhaled isoflurane by placing the head of the mouse into the nose cone attached to the isoflurane dispenser and cover the mouse (except the hindpaw designated for operation) with sterile Press’n Seal (Figure 2).


    Figure 2. Continuous inhalational anesthesia. The mouse was continuously anesthetized through a nose cone with 1.5%-2% isoflurane.

    For Steps 5-13 also see Video 1.
    Video 1. Plantar incision surgery. This video shows how the model of postoperative pain is made by making an incision through the plantar skin and flexor digitorum brevis muscle (This video was made at the Medical College of Wisconsin and was performed according to guidelines on Animal Care and approved by the Animal Research Ethics Board of the Medical College of Wisconsin under protocol #0383).

  5. Adhere the hindpaw to the surface by taping the toes down with surgical tape (Figure 3).


    Figure 3. Securing the hindpaw to surgical surface. The hindpaw was secured to the surface using surgical tape.

  6. Once the hindpaw is secured, apply a cotton swab of 75% ethanol followed by a new cotton swab of betadine. Repeat for a total of 3 applications each. 
  7. Measure 2 mm from the proximal edge of heel using a digital caliper and place a dot with a permanent marker at this location in the middle of the hindpaw. From the first dot, measure 5 mm towards the toes down the center of the hindpaw and place a second dot (Figure 4).


    Figure 4. Measurement of incision. Two dots were placed in the middle of the hindpaw, one 2 mm from the heel and the second 5 mm from the first dot.

  8. Check that the mouse is fully anesthetized by lightly pinching the most medial toe (most likely this toe could not be secured by the surgical tape) with the forceps. If the mouse flinches, wait until the mouse no longer reacts to the toe pinch before proceeding to Step 9. 
  9. Stabilize the hindpaw by placing the forceps on each side of the heel and make a longitudinal incision through the skin and fascia from the first dot to the second dot (Figure 5).


    Figure 5. Cutaneous incision. A 5 mm longitudinal incision was made with a No. 11 scalpel.

  10. Spread the skin away from the flexor digitorum brevis muscle with the forceps. Elevate the flexor digitorum brevis muscle by inserting one end of the curved forceps underneath the lateral edge of the flexor digitorum brevis muscle and pushing the forceps through to the medial side of the muscle (Figure 6).


    Figure 6. Elevation of flexor digitorum brevis muscle. Curved forceps were inserted under the flexor digitorum brevis muscle to elevate the muscle.

  11. Make a longitudinal incision with the scalpel through the entire belly of the muscle from the origin and insertion taking care not to sever the muscle completely from the origin and insertion, making sure to cut the belly of the muscle into two halves (Figure 7).


    Figure 7. Muscle incision. A longitudinal incision was made through the muscle belly of the elevated flexor digitorum brevis muscle from proximal to distal ends of the cutaneous incision.

  12. To suture the wound, remove the curved forceps from underneath the muscle and elevate the edges of the skin surrounding the wound with forceps. Close the wound by putting two sutures in the skin (but not muscle) approximately 2 mm apart using 5-0 nylon sutures and a hemostat (Figure 8).


    Figure 8. Cutaneous suturing. The skin was closed with two 5-0 nylon sutures.

  13. Apply a generous amount of bacitracin ointment to the wound using a cotton swab and place the mouse in the new cage located on the Heat Therapy Pump pad from Step 2.

Data analysis

Statistical significance was determined with GraphPad Prism 7 Software and graphs are shown as mean ± SEM. A two-way ANOVA with a Sidak post-hoc was used to determine statistical significance for mechanical and heat thresholds. A complete description of statistics used for analyzing mechanical and heat threshold behavioral data is provided in Cowie et al. (2018).

Notes

  1. It is important to perform the operation as quickly as possible to reduce the need for repeated anesthesia and any side effects from anesthesia. In our laboratory, it takes a trained surgeon approximately 5 min to perform this procedure.
  2. For a control, a sham surgery is performed by anesthetizing the mouse, sterilizing the hindpaw as in Step 5, and applying bacitracin ointment to the plantar hindpaw.
  3. If bleeding occurs during the procedure, apply pressure to the incision site with a cotton swab until the bleeding stops.
  4. Mice are housed together.
  5. No care is required after surgery except for monitoring of sutures. 
  6. If using mice past postoperative day 3, remove sutures on postoperative day 4. Mice that pulled out sutures before postoperative day 2 must be removed from the study due to poor wound closure.
  7. For consistent behavioral results, apply mechanical and heat stimuli to the medial-posterior aspect of the plantar hindpaw (Figure 9). This area is the least sensitive at baseline because the heel is weight bearing whereas other areas near the pads are more sensitive and variable in withdrawal threshold. Therefore, using the heel area that provides a consistently high baseline withdrawal threshold allows for the best detection of change due to incision (Brennan et al., 1996). Mice were acclimated for 1 h in Plexiglass chambers placed on either a mesh platform (mechanical threshold) or glass platform (heat threshold). Calibrated von Frey monofilaments (0.09 to 19.6 mN) were applied to the plantar hindpaw and the withdrawal threshold for each animal was calculated using the up-down method (Dixon, 1980; Chaplan et al., 1994). The Hargreaves assay was used to measure heat sensitivity as previously established (Hargreaves et al., 1988; Jackson et al., 1995; Barabas and Stucky, 2013; Cowie et al., 2018). Withdrawal latencies to a focused radiant heat source (IITC, Life Sciences Instruments) underneath the glass platform were measured 3 times and averaged for each mouse. A cutoff of 20 s was used to avoid injury. Examples of mechanical and heat hypersensitivity following incision are shown in Figure 10.


    Figure 9. Application of von Frey monofilament. An orange von Frey monofilament was applied to most sensitive location following incision.


    Figure 10. Mechanical and heat thresholds following incision. A. von Frey monofilaments was applied to most sensitive location following incision and the Dixon up-down method (Dixon, 1980) was used to determine mechanical threshold. B. The Hargreaves assay (Hargreaves et al., 1988) was used to measure the withdrawal threshold in response to a radiant heat source that was applied to the most sensitive location following incision. These data were modified from Cowie et al. (2018). Data shown as mean ± SEM, repeated-measures two-way ANOVA and Sidak post-hoc analysis, **P < 0.01 and ****P < 0.0001 sham versus incision. For (A) and (B), n = 8 male mice per group.

Acknowledgments

This protocol was adapted from established published procedures (Brennan et al., 1996; Pogatzki and Raja, 2003). This work was supported by the National Institute of Neurological Disorders and Stroke grants NS040538 and NS070711 to C.L.S and F31GM123778 to A.M.C. The Research and Education Component of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin provided partial support. The authors thank Timothy J Brennan, Ph.D., MD for his review of the manuscript.

Competing interests

The authors declare no competing financial or non-financial interests.

Ethics

All animal procedures were carried out in accordance with the National Institute of Health guidelines and approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin (AUA #0383).

References

  1. Barabas, M. E. and Stucky, C. L. (2013). TRPV1, but not TRPA1, in primary sensory neurons contributes to cutaneous incision-mediated hypersensitivity. Mol Pain 9: 9.
  2. Brennan, T. J. (2011). Pathophysiology of postoperative pain. Pain 152(3 Suppl): S33-40.
  3. Brennan, T. J., Vandermeulen, E. P. and Gebhart, G. F. (1996). Characterization of a rat model of incisional pain. Pain 64(3): 493-501.
  4. Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. and Yaksh, T. L. (1994). Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53(1): 55-63.
  5. Chapman, C. R. and Vierck, C. J. (2017). The transition of acute postoperative pain to chronic pain: An integrative overview of research on mechanisms. J Pain 18(4): 359 e1-359 e38.
  6. Cowie, A. M., Moehring, F., O'Hara, C. and Stucky, C. L. (2018). Optogenetic inhibition of CGRPα sensory neurons reveals their distinct roles in neuropathic and incisional pain. J Neurosci 38(25): 5807-5825.
  7. Dixon, W. J. (1980). Efficient analysis of experimental observations. Annu Rev Pharmacol Toxicol 20: 441-462.
  8. Fletcher, D., Stamer, U. M., Pogatzki-Zahn, E., Zaslansky, R., Tanase, N. V., Perruchoud, C., Kranke, P., Komann, M., Lehman, T. and Meissner, W. (2015). Chronic postsurgical pain in Europe: An observational study. Eur J Anaesthesiol 32(10): 725-734.
  9. Gan, T. J. (2017). Poorly controlled postoperative pain: prevalence, consequences, and prevention. J Pain Res 10: 2287-2298.
  10. Hargreaves, K., Dubner, R., Brown, F., Flores, C. and Joris, J. (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32(1): 77-88.
  11. Jackson, D. L., Graff, C. B., Richardson, J. D. and Hargreaves, K. M. (1995). Glutamate participates in the peripheral modulation of thermal hyperalgesia in rats. Eur J Pharmacol 284(3): 321-325.
  12. Pogatzki, E. M. and Raja, S. N. (2003). A mouse model of incisional pain. Anesthesiology 99(4): 1023-1027.
  13. Sen, S. and Bathini, P. (2015). Auditing analgesic use in post-operative setting in a teaching hospital. J Clin Diagn Res 9(4): FC01-04.
  14. Tan, W. H., Yu, J., Feaman, S., McAllister, J. M., Kahan, L. G., Quasebarth, M. A., Blatnik, J. A., Eagon, J. C., Awad, M. M. and Brunt, L. M. (2018). Opioid medication use in the surgical patient: An assessment of prescribing patterns and use. J Am Coll Surg 227(2): 203-211.
  15. Weiser, T. G., Regenbogen, S. E., Thompson, K. D., Haynes, A. B., Lipsitz, S. R., Berry, W. R. and Gawande, A. A. (2008). An estimation of the global volume of surgery: a modelling strategy based on available data. Lancet 372(9633): 139-144.
  16. Xu, J. and Brennan, T. J. (2010). Guarding pain and spontaneous activity of nociceptors after skin versus skin plus deep tissue incision. Anesthesiology 112(1): 153-164.

简介

术后疼痛非常虚弱,阻碍了恢复。 阿片类药物是用于急性术后疼痛的主要止痛药。 鉴于美国各地的阿片类药物成瘾和过量流行,非急需非阿片类药物疼痛治疗。 为了开发用于治疗术后疼痛的新型非阿片类药物疗法并确定这种疼痛的机制,已经建立了切口疼痛的啮齿动物模型。 本文的方案详细描述了如何创建根据已建立的方案改编的术后疼痛的小鼠模型。 这种术后疼痛模型经常使用,高度可重复,并导致外周和中枢神经系统改变。

【背景】术后疼痛是一个重要的全球性问题。每年大约有2.342亿人接受大手术(Weiser et al。,2008),大约80%的患者出现急性术后疼痛(Gan,2017)。其中,10%至50%的患者出现慢性疼痛,继续严重影响其生活质量(Chapman和Vierck,2017)。与发生慢性术后疼痛相关的因素之一,但不太可能的原因,是术后第一周出现急性疼痛的严重程度(Fletcher et al。,2015; Chapman和Vierck, 2017年)。阿片类药物是用于急性术后疼痛的主要止痛药(Sen和Bathini,2015; Tan et al。,2018)。鉴于阿片类药物流行,需要非阿片类药物疼痛治疗。因此,确定导致急性术后疼痛的机制对于开发术后疼痛的最佳疗法是必要的,这可能最终降低慢性术后疼痛的严重性和/或发生率。大鼠(Brennan et al。,1996)和小鼠(Pogatzki和Raja,2003)急性切口疼痛模型已被开发为临床前模型,以确定术后疼痛的分子,细胞和生理机制。 。然而,缺乏对术后疼痛的小鼠模型的详细描述。在这里,我们详细描述了需要切开皮肤和肌肉的术后疼痛的小鼠模型。皮肤和肌肉的切口最能模仿侵入性手术,导致剧烈的急性疼痛并导致慢性疼痛(Brennan,2011; Chapman和Vierck,2017)。此外,皮肤和肌肉的切口(约6天)会产生超敏反应,其持续时间比仅皮肤(~3天)切口模型长得多(Xu和Brennan,2010)。在该协议中,我们提供了从先前的方案(Brennan 等人,<1996; Pogatzki和Raja,2003)改编的详细的逐步方法,用于开发术后疼痛的小鼠模型。

关键字:术后疼痛, 炎症性疼痛, 超敏反应, 足底切口, 小鼠, 皮肤和肌肉切口

3 140 术后疼痛极易使人衰弱并阻碍康复。阿片类药物是用于急性术后疼痛的主要止痛药。鉴于全美的阿片类药物成瘾性严重和药物过量流行,迫切需要非阿片类药物止痛药。为了开发用于治疗术后疼痛的新颖的非阿片类药物疗法并确定这种疼痛的潜在机制,已建立了切开性疼痛的啮齿动物模型。本文中的协议详细描述了如何创建适用于既定协议的术后疼痛的小鼠模型。术后疼痛的这种模型是经常使用的,高度可复制的,并且会导致周围和中枢神经系统的改变。 【背景】术后疼痛是一个重大的全球性问题。每年大约有2.342亿人接受大手术(Weiser等人,2008年),约80%的患者经历了急性术后疼痛(Gan,2017年)。其中,约10%至50%的患者会发展为慢性疼痛,并继续严重影响其生活质量(Chapman and Vierck,2017)。与术后慢性疼痛发展相关的因素之一(但不太可能是原因)是术后第一周出现的急性疼痛的严重程度(Fletcher > et al。,2015; Chapman and Vierck, 2017)。阿片类药物是用于急性术后疼痛的主要止痛药(Sen和Bathini,2015年; Tan >等人,,2018年)。考虑到阿片类药物的流行,需要非阿片类药物的止痛药。因此,为开发可能最终降低慢性术后疼痛的严重程度和/或发生率的术后疼痛的最佳疗法,确定急性术后疼痛的基础机制是必要的。已将大鼠(Brennan等人,1996年)和小鼠(Pogatzki和Raja,2003年)的急性切开疼痛模型开发为临床前模型,以鉴定术后疼痛的分子,细胞和生理机制。但是,缺少对小鼠术后疼痛模型的详细描述。在这里,我们详细描述了需要切除皮肤和肌肉的术后疼痛的小鼠模型。皮肤和肌肉的切开最好模拟侵入性手术,该手术会引起剧烈的急性疼痛并导致慢性疼痛(Brennan,2011; Chapman and Vierck,2017)。此外,皮肤和肌肉的切口(〜6天)产生的超敏反应比仅皮肤的切口模型(〜3天)持续的时间更长(Xu和Brennan,2010)。在该协议中,我们提供了根据先前协议(Brennan等,1996; Pogatzki和Raja,2003)开发的详细,循序渐进的方法,用于开发术后疼痛的小鼠模型。

材料和试剂

  1. 不锈钢无菌11号手术刀片(World Precision Instruments,羽毛安全剃刀有限公司,目录号:504170)
  2. 无菌5-0尼龙手术缝合线(AD Surgical,Unify,货号:S-N518R13)
  3. 手术胶带(3M,Transpore,货号:1527-0)
  4. 棉签(VWR,临界棉签,货号:89031-270)
  5. Glad Press'n Seal(SAI输液技术,Glad,目录号:PSS-70)
  6. 无菌丁腈手套(Kimberly-Clark Professional,Kimtech Pure,目录号:HC61170)
  7. 培养皿[VWR,14.5和9 663161,Greiner Bio-One,目录号:82050-912(小)和82050-600(大)]
  8. 无菌纱布(Alleded Medical,Ardes,目录号:GA441221)
  9. Sharpie特细永久标记(Staples,Sharpie,目录号:37001)
  10. 8-16周大的C57BL / 6J小鼠(JAX,目录号:000664)
  11. 杆菌肽锌软膏(Fougera Pharmaceuticals Inc,目录号:0168-0011-04)
  12. 异氟烷(Clipper Distribution Company LLC。,Phoenix,目录号:0010250)
  13. 75%的乙醇(Fisher Scientific,Decon Laboratories,Inc.,目录号:22-281-562)
  14. 手术擦洗7.5%聚维酮碘(贝他定,兽医,目录号:67618-154-01)
  15. 眼部润滑剂(Patterson兽医,眼科Optixcare,目录号:07-893-2779)

设备

  1. 1,000 ml烧杯(VWR,PYREX,货号:13912-284)
  2. #55 Dumostar镊子(Fine Science Tools,Dumont,目录号:11295-51)
  3. 手术刀手柄(精细科学工具,目录号:10003-12)
  4. 虹膜镊子,10厘米,弯曲,锯齿状(World Precision Instruments,目录号:15915)
  5. Halsted蚊子止血钳,12.5厘米,直式(World Precision Instruments,目录号:15920-G)
  6. Vannas剪刀,弯曲的8厘米(World Precision Instruments,目录号:14122)
  7. 小动物手术板(Braintree Scientific,Inc.,CD +,目录号:ACD 014)
  8. 异氟烷分配器(Highland Medical Equipment,Drager,目录号:16-7001)
  9. 滑动顶部异氟烷感应室(肯特科学公司,目录号:VetFlo-0530LG)
  10. 带垫热疗泵(Adroit Medical Systems,目录号:HTP-1500)
  11. 等温垫(Braintree Scientific,Inc.,Deltaphase,目录号:DPIP)
  12. 数字卡尺(VWR,目录号:62379-531)
  13. Steri 250珠灭菌器珠浴(实验室无限,西蒙·凯勒有限公司,目录号:4AJ-6286283)
  14. 微波炉(Emerson,1,000 W,目录号:B007Q45CIS)
  15. 装有Aspen Sani Chips ®(PJ Murphy Forest and Products,Sani Chips ®)的家用笼

软件

  1. GraphPad棱镜7

程序

  1. 用乙醇对手术区域进行消毒,并在至少325°C的珠浴中对工具进行消毒30 s。将无菌工具,缝线和刀片放在无菌纱布上。
  2. 将热疗法泵垫加热至41°C,然后在微波炉中将等温垫加热1分钟。将新的干净家用笼子放在热疗泵垫上。将实验室用纸巾放在温暖的等温垫上,并盖上无菌Press'n Seal和手术台。
  3. 为了引起麻醉,将鼠标放在装有1 ml异氟烷的纸巾上的烧杯腔中(图1),并等待大约30 s或直到鼠标不再正确。或者,可以使用异氟烷分配器随附的异氟烷诱导室来诱导麻醉。


    图1.简短的吸入麻醉。通过在室内用1 ml异氟烷吸入将小鼠短暂麻醉。

  4. 从异氟烷腔中快速移出鼠标,并用棉签向每只眼睛涂抹眼油。通过将小鼠的头部放入与异氟烷分配器相连的鼻锥中,用1.5%-2%吸入的异氟烷进一步麻醉小鼠,并用无菌Press'n Seal盖住小鼠(指定用于手术的后爪除外)(图2) 。


    图2.持续吸入麻醉。小鼠通过鼻锥和1.5%-2%异氟烷连续麻醉。

    有关步骤5-13 ,另请参见视频1。
    视频1.足底切口手术。该视频显示了如何通过对足底皮肤和指趾短屈肌进行切开来制作术后疼痛模型(该视频由威斯康星州医学院和美国是根据动物保健指南进行的,并由威斯康星州医学院的动物研究伦理委员会根据协议#0383批准。

  5. 用手术胶带将脚趾向下敲打,将后爪粘在表面上(图3)。


    图3.将后爪固定到手术表面。用手术胶带将后爪固定在表面上。

  6. 固定后爪后,先用75%乙醇的棉签擦拭,再用新的甜菜碱棉签擦拭。重复总共3个应用程序。
  7. 使用数字卡尺从脚后跟的近端边缘测量2毫米,并在后爪中间的此位置放置一个带有永久标记的点。从第一个点开始,朝后脚中央的脚趾方向测量5毫米,然后放置第二个点(图4)。


    图4.切口的测量。在后爪中间放置两个点,距离脚后跟一个2毫米,另一个距离第一个点5毫米。

  8. 用镊子轻轻捏住最内侧的脚趾(最有可能无法用手术胶带固定该脚趾),以检查鼠标是否已完全麻醉。如果鼠标退缩,请等到鼠标不再对脚趾捏起反应后再继续执行步骤9。
  9. 通过将镊子放在脚跟的每一侧来稳定后爪,并从第一个点到第二个点在皮肤和筋膜上进行纵向切口(图5)。


    图5.皮肤切口。用11号手术刀切开5毫米的纵向切口。

  10. 用镊子使皮肤远离屈指短肌。通过将弯曲镊子的一端插入屈指短肌的外侧边缘下方,并将镊子推入肌肉的内侧,来提升屈短指短肌。

    图6.屈短指短肌高程。将弯曲的镊子插入短屈指短肌下方以升高肌肉。

  11. 用手术刀从源头和插入处穿过整个腹部进行纵向切口,注意不要从源头和插入处完全切断肌肉,确保将腹部切成两半(图7)。


    图7.肌肉切口。从皮肤切口的近端到远端,通过高位屈指短肌的肌肉腹部进行纵向切口。

  12. 要缝合伤口,请从肌肉下方取下弯曲的镊子,并用镊子抬高伤口周围的皮肤边缘。通过使用5-0尼龙缝线和止血器在皮肤(但不包括肌肉)中以大约2 mm的间隔放置两条缝线来闭合伤口(图8)。


    图8.皮肤缝合。用两个5-0尼龙缝线闭合皮肤。

  13. 用棉签在伤口上涂抹大量杆菌肽软膏,然后将鼠标放在步骤2中位于热疗泵垫上的新笼子中。

数据分析

用GraphPad Prism 7软件确定统计显着性,并且图显示为平均值±SEM。具有Sidak post hoc的双向ANOVA用于确定机械和热阈值的统计显着性。Cowie等人(2018)提供了用于分析机械和热阈值行为数据的统计信息的完整说明。

笔记

  1. 重要的是尽快执行手术,以减少重复麻醉的必要性以及麻醉带来的任何副作用。在我们的实验室中,需要经过培训的外科医生大约5分钟才能执行此过程。
  2. 作为对照,通过如下步骤进行假手术:对小鼠进行麻醉,如步骤5中那样对后爪进行消毒,并且将杆菌肽软膏施用于足底后爪。
  3. 如果在操作过程中发生出血,请用棉签对切口部位施加压力,直到出血停止为止。
  4. 小鼠被安置在一起。
  5. 手术后无需进行任何护理,除了可以监控缝合线。
  6. 如果在术后第3天之后使用小鼠,则在术后第4天去除缝线。由于伤口闭合不良,必须在术后2天之前拔出缝线的小鼠从研究中移除。
  7. 为获得一致的行为结果,请对足后爪的内侧-后侧施加机械刺激和热刺激(图9)。该区域在基线时最不敏感,因为脚后跟负重,而垫附近的其他区域更敏感,并且退缩阈值可变。因此,使用提供始终如一的高基线撤回阈值的脚后跟区域可以最好地检测出切口造成的变化(Brennan等,1996)。使小鼠在置于网状平台(机械阈值)或玻璃平台(热阈值)上的有机玻璃室内适应1小时。将校准的von Frey单丝(0.09至19.6 mN)应用于足后爪,并使用上下方法计算每只动物的撤离阈值(Dixon,1980; Chaplan et al。,1994)。 。如先前建立的那样,使用Hargreaves测定法来测量热敏感性(Hargreaves等人,1988年;杰克逊等人,1995年; Barabas和Stucky,2013年; Cowie等。 > et al。,2018年)。对玻璃平台下方聚焦辐射热源(IITC,生命科学仪器)的撤回潜伏期进行了3次测量,并对每只小鼠取平均值。截止时间为20 s,以避免受伤。切口后的机械和热超敏反应示例如图10所示。


    图9. von Frey单丝的应用。在切口后将橙色的von Frey单丝应用于最敏感的位置。


    图10.切口后的机械和热阈值。 A。切口后将von Frey单丝应用于最敏感的部位,并使用Dixon上下法(Dixon,1980)确定机械阈值。B. Hargreaves测定法(Hargreaves等人,1988年)用于测量切开后响应于辐射热源的辐射热源的撤离阈值。这些数据是从Cowie等人(2018)修改而来的。数据显示为平均值±SEM,重复测量的两次方差分析和Sidak事后分析,** > P &lt; 0.01和**** > P &lt; 假手术vs切开手术0.0001 对于(A)和(B),每组n = 8只雄性小鼠。

致谢

该协议改编自已公开的程序(Brennan等,1996; Pogatzki和Raja,2003)。这项工作得到了美国国家神经疾病研究所的支持,中风授予了CLS NS040538和NS070711,授予AMC授予了F31GM123778。威斯康星医学院的“提高健康的威斯康星基金会”的研究和教育部分提供了部分支持。作者感谢Timothy J Brennan博士对手稿的审阅。

利益争夺

作者声明没有任何竞争性的金融或非金融利益。

伦理

所有动物程序均按照美国国立卫生研究院的指导原则进行,并由威斯康星州医学院的机构动物护理和使用委员会(AUA#0383)批准。

参考文献

  1. 缅因州巴拉巴斯(Barabas)和缅因州斯塔基(Stucky)(2013)。初级感觉神经元中的TRPV1而非TRPA1有助于皮肤切口介导的超敏反应。 >摩尔痛 9:9。
  2. 布伦南(TJ)(2011)。术后疼痛的病理生理学。 >疼痛 152(3补充) ):S33-40。
  3. Brennan,TJ,Vandermeulen,EP和Gebhart,GF(1996)。描述了一种大鼠切入性疼痛模型的特征。 >疼痛 64(3):493-501。
  4. Chaplan,SR,Bach,FW,Pogrel,JW,Chung,JM和Yaksh,TL(1994)。大鼠足部触觉异常性疼痛的定量评估。 > J Neurosci Methods < 53(1):55-63。
  5. Chapman,CR和Vierck,CJ(2017)。急性术后疼痛向慢性疼痛的过渡:机制研究的综合综述。 > J痛苦 18(4):359 e351-359 e338。
  6. Cowie,AM,Moehring,F.,O'Hara,C.和Stucky,CL(2018)。对CGRPα感觉神经元的光遗传学抑制揭示了它们在神经性和切开性疼痛中的独特作用。 J Neurosci 38(25):5807-5825。
  7. Dixon,WJ(1980)。有效分析实验观察结果。 > Annu Rev Pharmacol Toxicol 20:441-462。
  8. Fletcher,D.,Stamer,UM,Pogatzki-Zahn,E.,Zaslansky,R.,Tanase,NV,Perruchoud,C.,Kranke,P.,Komann,M.,Lehman,T.和Meissner,W.( 2015)。欧洲的慢性术后疼痛:一项观察性研究。 > Eur J Anaesthesiol < 32(10):725-734。
  9. Gan TJ(2017)。术后疼痛控制不佳:患病率,后果和预防措施。 > J痛Res 10:2287-2298。
  10. Hargreaves,K.,Dubner,R.,Brown,F.,Flores,C。和Joris,J。(1988)。一种新的敏感方法,用于测量皮肤痛觉过敏的热伤害感受。 >疼痛 32(1):77-88。
  11. 杰克逊(DL),格拉夫(Craf),世邦魏理仕(CB),理查森(Richardson),法学博士和哈格里夫斯(KM)(1995)。谷氨酸盐参与大鼠热痛觉过敏的外周调节。 > Eur J Pharmacol 284(3):321-325。
  12. Pogatzki,EM和Raja,SN(2003)。一种切口疼痛的小鼠模型。 >麻醉学 99( 4):1023-1027。
  13. Sen和S.Bathini,P.(2015年)。在教学医院的术后环境中对止痛药进行审核。 > J临床诊断研究 9(4):FC01-04。
  14. Tan,WH,Yu,J.,Feaman,S.,McAllister,JM,Kahan,LG,Quasebarth,MA,Blatnik,JA,Eagon,JC,Awad,MM和Brunt,LM(2018)。在外科手术患者中使用阿片类药物治疗:处方和使用方法的评估。 J Am Coll Surg 227(2):203-211。
  15. Weiser,TG,Regenbogen,SE,Thompson,KD,Haynes,AB,Lipsitz,SR,Berry,WR和Gawande,AA(2008)。对全球手术量的估算:一种基于可用数据的建模策略。 > Lancet 372(9633):139-144。
  16. Xu J.和Tren Brennan(2010)。保护皮肤和皮肤以及深层组织切口后伤害感受器的疼痛和自发活动。 麻醉学 112(1):153-164。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
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: © 2019 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. Cowie, A. M. and Stucky, C. L. (2019). A Mouse Model of Postoperative Pain. Bio-protocol 9(2): e3140. DOI: 10.21769/BioProtoc.3140.
  2. Cowie, A. M., Moehring, F., O'Hara, C. and Stucky, C. L. (2018). Optogenetic inhibition of CGRPα sensory neurons reveals their distinct roles in neuropathic and incisional pain. J Neurosci 38(25): 5807-5825.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

当遇到任何问题时,强烈推荐您通过上传图片的形式提交相关数据。