在手术伤口的痛觉及炎症介质的收集和测量

Biology

ERRATUM NOTICE

Cite this Article

Copy Citation | Download Citations

Carvalho, B., Clark, D. J., Yeomans, D., Angst, M. S. Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds . J. Vis. Exp. (20), e962, doi:10.3791/962 (2008).

Please note that all translations are automatically generated.

Click here for the english version. For other languages click here.

Abstract

这项研究的目的是测试在手术部位的炎症和伤害性的生化介质的收集和测量的可行性,评估伤口和血清水平之间的关系,并确定介质的释放,疼痛和镇痛消费后剖宫产之间的任何关联交付。二十经历选修剖宫产脊髓麻醉的健康妇女参加。 1,6,24,和48小时后剖宫产伤口渗出液和血清介质,疼痛评分和止痛剂的消费。在创面渗液,调解员19 20出可靠的检测,包括IL -1β,IL - 2,IL - 4,IL - 6,IL - 7,IL - 8,IL - 10,IL - 12,IL - 13,白细胞介素体育伤口PG - E2和各种细胞因子-17,肿瘤坏死因子,INFγ,G - CSF,GM - CSF,MCP - 1和MIP -1β,神经生长因子(NGF),前列腺素E2(PG - E2的)和物质见顶早,而神经生长因子表现出更多的延迟​​释放。有伤口和血清细胞因子的浓度随时间变化的轮廓之间没有相关性。这项研究表明,在手术伤口的痛觉和炎症介质,在特定时间点的收集和测量的可行性。伤口和血清之间缺乏显着相关性,强调本地化的病状,如果要研究确定站点的具体发布的重要性。

Protocol

伤害性和炎症的生化调解员收集

  1. ON - Q ® PainBuster ®止痛系统插入皮下层由手术队前伤口缝合。该系统持续皮下伤口在2毫升/小时的速度提供生理盐水(或局部麻醉)
  2. 一个三路阀被纳入这个系统允许在指定的时间点伤口渗出的愿望。
  3. 在指定的时间点协议(例如,1,6,24,和剖宫产分娩后48小时),伤口渗出液1毫升撤回到聚乙烯杯含30μL蛋白酶抑制剂。
  4. 在相同的时间间隔,10毫升的血液被收集成为绿色顶级采血含锂肝素管。然后加入300μL蛋白酶抑制剂的血液样本。
  5. 在1小时收集,样品置于冰上,并在10分钟3000转离心。
  6. 血清及创面supranate被删除并放置在一个标准的离心管,并储存在-20 ° C

含量分析

  1. 一旦所有样品的采集,他们是解冻和分析,在同一时间。
  2. 细胞因子,然后用A17复珠阵列免疫板。多重免疫检测技术可以在50μl和产生的结果与用ELISA法获得的相比体积小的体液样本化验100分析物。这个板块是能够测量1ß白细胞介素(IL -1β),白细胞介素2(IL - 2),白细胞介素4(IL - 4),白细胞介素5(IL - 5),白细胞介素6(IL - 6),白细胞介素7(IL -7),白细胞介素8(IL - 8),白细胞介素(IL - 10),白细胞介素12(IL - 12),白细胞介素13(IL - 13),白细胞介素17(IL - 17),肿瘤坏死因子α(TNFα) ,干扰素α(INFα),粒细胞集落刺激因子(G - CSF的),粒细胞 - 巨噬细胞集落刺激因子(GM - CSF的),单核细胞趋化蛋白1(MCP - 1)和巨噬细胞炎性蛋白(MIP -1β)。
  3. 神经生长因子NGF抗体DY256测量与生物复杂胺耦合试剂盒的援助增加17的plex板。
  4. 每一次测量是在重复,并根据制造商的规范。每个分析物的标准曲线所产生的使用参考由厂家提供的分析物浓度为0.20,0.78,3.13,12.5,50,200,800,3200皮克/毫升加一个零标准(生理盐水)。在每次运行的标准曲线和样品浓度与生物复杂的管理器软件计算。
  5. 前列腺素E2和P物质的测量,使用一个高灵敏度ELISA法根据制造商的规范,每个检测是在重复执行工具箱。

    图1
    图1

    图1:各种亲和抗炎细胞因子(1ß白细胞介素(IL -1β),白细胞介素2(IL - 2),白细胞介素4(IL - 4),白细胞介素5(IL - 5),白细胞介素的渗出液和血清6(IL - 6),白细胞介素7(IL - 7),白细胞介素8(IL - 8),白细胞介素(IL - 10),白细胞介素12(IL - 12),白细胞介素13(IL - 13),白细胞介素17(IL -17),肿瘤坏死因子α(TNF -α),干扰素γ(INFγ),粒细胞集落刺激因子(G - CSF的),粒细胞 - 巨噬细胞集落刺激因子(GM - CSF的),单核细胞趋化蛋白1(MCP - 1 )和巨噬细胞炎性蛋白(MIP -1β),神经生长因子(NGF),前列腺素E2(PG - E2的)和P物质(SP)的水平(pg / mL的)在基线测定,6至24小时后剖宫产。

Subscription Required. Please recommend JoVE to your librarian.

Discussion

ON - Q ® PainBuster ®止痛系统应横跨整个皮下层切口插入前伤口缝合。这有利于通过在指定的时间间隔三路阀的愿望。 ON - Q ®系统不断传递到伤口的生理盐水皮下注射2毫升/小时的速度这可以防止导管凝血,提高了系统的可靠性产生渗出液样本。抽吸渗液如果困难(约5%的情况下),考虑改变主体的位置(例如,坐在病人最多,或让它们平铺),推伤口上方轻轻,使用一个0.5 -1毫升正常生理盐水冲洗或撤回导管1-2厘米。

Subscription Required. Please recommend JoVE to your librarian.

Acknowledgements

卡瓦略博士的工作是支持由妇女健康研究资助妇女的健康和国家的儿童健康和人类发展的美国国立卫生研究院(5K12 HD043452)研究所的研究办公室的跨学科人才招聘大厦。焦虑医生收到用品(ON - Q ® PainBuster ®运算后疼痛缓解系统)和资金进行从我流(CA)的森林湖,生化分析。

Materials

Name Company Catalog Number Comments
On-Q® PainBuster® Post-Op Pain Relief System I-Flow, Lake Forest, CA
Complete proteinase inhibitor Roche Group
17-multiplex bead immunoassay Bio-PlexTM plate Bio-Rad
Bio-Plex amine coupling kit Bio-Rad
The NGF antibody DY256 R&D Systems
Prostaglandin E2 and substance P ELISA Kits Assay Designs

DOWNLOAD MATERIALS LIST

References

  1. Elshal, M. F., McCoy, J. P. Multiplex bead array assays: performance evaluation and comparison of sensitivity to ELISA. Methods. 38, 317-323 (2006).
  2. Heijmans-Antonissen, C., Wesseldijk, F., Munnikes, R. J., Huygen, F. J., van der Meijden, P., Hop, W. C., Hooijkaas, H., Zijlstra, F. J. Multiplex bead array assay for detection of 25 soluble cytokines in blister fluid of patients with complex regional pain syndrome type 1. Mediators Inflamm.. 28398, 1-8 (2006).
  3. Buvanendran, A., Kroin, J. S., Berger, R. A., Hallab, N. J., Saha, C., Negrescu, C., Moric, M., Caicedo, M. S., Tuman, K. J. Upregulation of prostaglandin E2 and interleukins in the central nervous system and peripheral tissue during and after surgery in humans. Anesthesiology. 104, 403-410 (2000).
  4. Holzheimer, R. G., Steinmetz, W. Local and systemic concentrations of pro- and anti-inflammatory cytokines in human wounds. Eur J Med Res. 5, 347-355 (2000).

Erratum

Formal Correction: Erratum: Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds
Posted by JoVE Editors on 03/07/2012. Citeable Link.

A correction was made to: Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds. A key reference was excluded.

A fifth reference:

5. Carvalho, B., Clark, D. J. & Angst, M. S. Local and Systemic Release of Cytokines, Nerve Growth Factor, Prostaglandin E2, and Substance P in Incisional Wounds and Serum Following Cesarean Delivery. The Journal of Pain : official journal of the American Pain Society 9 (7), 650-657 (2008).

was added. The abstract was updated to :

We describe a methodology by which we are able to collect and measure inflammatory and nociceptive biochemical mediators at the surgical wound site. Collecting site-specific biochemical markers allows us to evaluate the relationship between surgical wound and serum levels; determine any associations between mediator release, pain and analgesic consumption; and evaluate the effect of systemic and peripheral drug administration on surgical wound biochemistry.

This methodology has been applied to healthy women undergoing elective cesarean delivery with spinal anesthesia. Wound exudate and serum mediators, in conjunction with pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean delivery. Biochemical mediators that were detected included IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNFα, INFγ, G-CSF, GM-CSF, MCP-1 and MIP-1β, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. We found no correlations between wound and serum cytokines concentrations or time-release profiles (J Pain. 2008 Jul 9(7):650-7). This article describes and demonstrates the feasibility of collecting and assaying nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between serum and wound levels shows the importance of determining site-specific release if surgical wounds and localized pathologies are to be studied.

from

The objectives of this study were to test the feasibility of collecting and measuring inflammatory and nociceptive biochemical mediators at the surgical site; to evaluate the relationship between wound and serum levels; and to determine any associations between mediator release, pain and analgesic consumption post-cesarean delivery. Twenty healthy women undergoing elective cesarean delivery with spinal anesthesia were enrolled. Wound exudate and serum mediators, pain scores and analgesics consumption were measured at 1, 6, 24, and 48 hours post-cesarean. In wound exudate, 19 out of 20 mediators were reliably detected including IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, TNFα, INFγ, G-CSF, GM-CSF, MCP-1 and MIP-1β, nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P. Wound PG-E2 and various cytokines peaked early, whereas NGF showed a more delayed release. There were no correlations between the concentration versus time profile of wound and serum cytokines. This study demonstrates the feasibility of collecting and measuring nociceptive and inflammatory mediators in surgical wounds at specific time points. The lack of significant correlations between wound and serum levels emphasizes the importance of determining site-specific release if localized pathologies are to be studied.

Comments

1 Comment

  1. The technique appears robust, even if intuitive. However, as shown in the video paper, the data are collected in the presence of superfusion with local anaesthetic solution (in this case bupivacaine) which is very likely to modify the response and the local concentrations of mediators both as a direct pharmacological action of the local anaesthetic as well as through the fluid diluent effect. Moreover, adsoprtion of the various substances by the medical plastics would need evaluation. Hence a lot of control work needs to be done so that the numerical results obtained can be interpreted correctly. Perhaps the data are semiquantitative at best.

    Reply
    Posted by: Anonymous
    March 4, 2009 - 6:37 PM

Post a Question / Comment / Request

You must be signed in to post a comment. Please or create an account.

Usage Statistics