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Collecting and Measuring Nociceptive and Inflammatory Mediators in Surgical Wounds

1, 1, 1, 1

1Department of Anesthesiology, Stanford University School of Medicine

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    This article has been retracted at the request of the authors due to duplication of text in J Pain (Carvalho B, Clark DJ, Angst MS (2008) Local and Systemic Release of Cytokines, Nerve Growth Factor, Prostaglandin E2, and Substance P in Incisional Wounds and Serum Following Cesarean Delivery. J Pain 9: 650-657.) due to an honest mistake of the authors.

    Summary

    A technique to collect and measure surgical wound biochemical mediators at specific time points.

    Date Published: 10/29/2008, Issue 20; doi: 10.3791/962

    Cite this Article

    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).

    Abstract

    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.

    Protocol

    Nociceptive and inflammatory biochemical mediator collection

    1. The On-Q® PainBuster® Pain Relief System is inserted into the subcutaneous layer by the surgical team just prior to wound closure. The system continuously delivers normal saline (or local anesthetic) subcutaneously into the wound at a rate of 2 ml/h.
    2. A three-way stopcock is incorporated into this system to allow aspiration of wound exudate at specified time points.
    3. At time points specified by the protocol (e.g., 1, 6, 24, and 48 hours after cesarean delivery), 1 ml of wound exudate is withdrawn into a polyethylene cup containing 30 μl of proteinase inhibitor.
    4. At the same time intervals 10 ml of blood is collected into a green top blood collection tube containing lithium heparin. 300 μl of proteinase inhibitor is then added to the blood samples.
    5. Within 1 hour of collection, samples are put on ice and centrifuged at 3000 rpm for 10 min.
    6. The serum and wound supranate is removed and place in a standard microcentrifuge tube and stored at -20°C

    Assay analysis

    1. Once all samples are collected, they are thawed and analyzed at the same time.
    2. Cytokines are then measured using a17-multiplex bead array immunoassay plate. Multiplex immuno-assay technology allows assaying up to 100 analytes in body-fluid samples as small in volume as 50 μl and produce results comparable with those obtained with ELISA This plate is capable of measuring interleukin 1ß (IL-1ß), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin (IL-10), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 17 (IL-17), tumor necrosis factor α (TNFα), interferon α (INFα), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 (MIP-1β).
    3. Nerve growth factor is measured with NGF antibody DY256 by adding it to the 17-plex plate with aid of the Bio-Plex amine coupling kit.
    4. Each measurement is made in duplicates and according to the manufacturer's specification. Standard curves for each analyte are generated by using the reference analytes supplied by the manufacturers at concentrations of 0.20, 0.78, 3.13, 12.5, 50, 200, 800, 3200 pg/ml plus a zero standard (normal saline only). Standard curves are included in each run and sample concentrations were calculated with Bio-Plex Manager software.
    5. Prostaglandin E2 and substance P are measured using a highly sensitivity ELISA Kits Each assay is performed in duplicate according to the manufacturer’s specification.

      Figure 1
      Figure 1

      Figure 1: Exudate and serum levels of various pro- and anti-inflammatory cytokines (interleukin 1ß (IL-1ß), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin (IL-10), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 17 (IL-17), tumor necrosis factor α (TNF-α), interferon γ (INFγ), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 (MIP-1ß), nerve growth factor (NGF), prostaglandin E2 (PG-E2) and substance P (SP) levels (pg/mL) measured at baseline, 6 and 24 hours post-cesarean delivery.

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    Discussion

    The On-Q® PainBuster® Pain Relief System should be inserted across the entire incision in subcutaneous layer just prior to wound closure. This facilitates aspiration via the three-way stopcock at the specified time intervals. The On-Q® system continuously delivers normal saline subcutaneously into the wound at a rate of 2 ml/h. This prevents the catheter clotting and improves the reliability of the system to produce exudate samples. If aspiration of exudate is difficult (approximately 5% of cases), consider changing the subject's position (e.g., sitting the patient up or lying them flat), pushing gently above the wound, using a 0.5-1ml normal saline flush or withdrawing the catheter 1-2 cm.

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    Disclosures

    The authors have nothing to disclose.

    Acknowledgements

    Dr. Carvalho’s work is supported by a Building Interdisciplinary Careers in Women's Health research grant from the Office of Research on Women's Health and National Institute of Child Health and Human Development of the National Institutes of Health (5K12 HD043452). Dr Angst received supplies (the On-Q® PainBuster® Post-Op Pain Relief System) and funding for the conducting the biochemical assays from I-Flow (Lake Forest, CA).

    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

    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

    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: AnonymousMarch 4, 2009, 6:37 PM

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