Name: Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators (Video) | JoVE Journal
Uploaded: 2008-12-01T00:00:00.000+00:00
Duration: 8 min 54 s
Description: Learn microdialysis catheter placement for tissue-specific collection of interstitial fluid from inflamed human skin. This method enables measurement of nociceptive and inflammatory mediators in pain research.

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5 minutes

Induction of experimental inflammation:
<ol>
<li>A first-degree sunburn is induced in non-tanned skin of a thigh with aid of a calibrated, ultraviolet B source (Saalmann Multitester SBB LT 499, Saalmann GmbH, Hereford, Germany). The sunburn is induced 24 hours before starting a microdialysis experiment by applying three times the amount of UVB light necessary to cause complete reddening of exposed skin, or three times the minimal erythemal dose (MED). The MED is determined in each subject before study initiation. Accurate delivery of energy is assured with a UV-meter (RM 12, Dr. Grobel UV-Electronic GmbH, Ettlingen, Germany).</li>
</ol>
Catheter placement and sample collection:
<ol>
<li>Prepare a staging area with sterile gloves, 1 sterile needle holder, 1cc luer tip syringe with a 23 gauge (G) needle, 1cc syringe with a 30G needle, 1% lidocaine, alcohol swabs, 2x2 sterile gauze pads, microdialysis catheters attached at one end to a 4cm 23G needle (Dermal Dialysis, Erlangen, Germany), and 1.5 ml Microcent collection tubes (E&amp;K Scientific, Santa Clara, CA.) with 4&micro;l protease inhibitor solution per 100&micro;l sample on dry ice. protease inhibitor solution is made per manufacturer&rsquo;s instruction using protease inhibitor cocktail tablets (Roche Diagnostics, GmbH, Mannheim, Germany).</li>
<li>Prepare a 1% human albumin solution in lactated ringer&rsquo;s solution and draw it into the 1cc luer tip syringe, making sure there are NO air bubbles remaining in the syringe.</li>
<li>Load the syringe with the 1% human albumin-enriched ringer&rsquo;s lactate solution into the cradle (microdialysis multi-syringe rack, Harvard Apparatus, South Natick, MA) mounted onto a high precision infusion pump (Pump 22, Harvard Apparatus, South Natick, MA). Make certain that the bar of the infusion pump driving the syringe is in full contact with the plunger of the syringe. Set the pump rate after confirming that the syringe diameter has been accurately entered into the pump. Commonly used infusion rates range between 2 to 4&micro;l/min.</li>
<li>Define the area you plan to collect your sample from. When sampling from an artificially created inflamed area, such as the sunburn described above, plan on inserting the catheter at the edge of the sunburn.</li>
<li>Clean a small area at opposite edges of the lesion, or site you intend to collect your sample from, with alcohol. These cleaned areas will be the intended insertion and exit points for the catheter. Inject just enough lidocaine intra-cutaneously (evidenced by blanching of the skin on injection) at these sites to create a very small bleb (maximum of 2mm). Injection of such limited amount of intra-cutaneous lidocaine at the entry and exit points of the microdialysis catheter should not alter the results of the bioassay but does make the insertion of the catheter significantly less painful for study subjects.</li>
<li>Using aseptic techniques, insert the microdialysis catheters with aid of attached 23G needle over the length of the explored skin area. The depth of insertion is at the interface between cutis and subcutis. Insertion at an adequate depth can be verified optically. As the needle and the attached catheter are moved forward the needle should cause a little up-folding of the skin as well as some blanching of the skin. No bleeding should occur. If bleeding does occur this indicates that the needle is located deeper than intended and the depth of insertion has to be adjusted. Once the needle has been pushed through the exit point of the skin, flush the catheters with 1% albumin enriched ringer&rsquo;s lactate solution until you see droplets along the length of the catheter. This is done by carefully inserting the needle end of a syringe (23G needle) into the distal end of the microdialysis catheter, and slowly injecting solution.</li>
<li>After flushing the catheter pull gently on the needle to feed the attached catheter through the skin. Gentle handling is required because the catheters are small-diameter, and consequently fragile. Pull the catheter until it is completely threaded through the skin, i.e., the base of the larger plastic tubing embedding the distal end of the catheter touches the skin at the entry point. As mentioned above, little to no bleeding at the end of the procedure confirms optimal catheter placement.</li>
<li>Attach the distal end of the catheter to the tip of the 23G needle of the syringe resting in the rack of the infusion pump. Care is required to avoid puncturing the needle through the wall of the catheter, which would cause potential leakage.</li>
<li>Cut the needle off the proximal end of the catheter.</li>
<li>Using &frac14;-inch pink tape, attach the prepared 1.5ml Microcent collection tube at the skin close to the exit site of the catheter. Place the tip of the catheter into the collection tube to allow collection of microdialysate. Start the perfusion pump at predetermined rate and intermittently check that delivered and collected volume match up. In case the volumes do not match up, leakage from the skin entry site should be excluded (can sometimes be resolved by pulling the part of the catheter that is embedded by the plastic tubing more tightly onto the skin entry site). Alternatively, a catheter may need to be replaced if delivered and collected volumes do not match up.</li>
<li>Once sampling is complete, the samples are placed on dry ice until storage at -80&ordm;C.</li>
</ol>

<ol><li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation%2C+noxious+stimulation+and+administration+of+a+COX-inhibitor%3A+A+microdialysis+study%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation and administration of a COX-inhibitor: A microdialysis study.</a> Pain. 139, 15-27 (2008).</li>
</ol>

The authors have nothing to disclose.

The combined use of microdialysis techniques and multiplex immunoassay technology is a valuable real-time, in-vivo human bioassay providing insight into biochemical events in tissue of interest. Combined with behavioral tests such as pain tests, the method allows studying the complex interactions of biochemicals mediating pain and inflammation. The method is promising for 1) identifying tissue specific biomarkers mediating pain and inflammation, 2) providing mechanistic insight into the pathology of inflammatory and chro...

<table><tbody><tr><td>Microdialysis catheter</td><td>Tool</td><td>Dermal Dialysis</td><td>NA</td><td></td></tr><tr><td>Pump 22</td><td>Tool</td><td>Harvard Apparatus</td><td>552219</td><td></td></tr><tr><td>Saalmann Multitester SBB LT 400</td><td>Tool</td><td>Saalmann GmbH</td><td>NA</td><td></td></tr><tr><td>RM-12 IP-65 Radiometer</td><td>Tool</td><td>Dr. Grobel UV Elektronik GmbH</td><td>82120000/81112100</td><td></td></tr><tr><td>Complete Proteinase Inhibitor</td><td>Reagent</td><td>Roche Group</td><td>11836145001</td><td></td></tr><tr><td>Microcent tube</td><td>Tool</td><td>E&amp;amp;K Scientific</td><td>380150-X</td><td></td></tr><tr><td>Albumin 5%</td><td>Reagent</td><td>ZLB Behring</td><td>NA</td><td></td></tr><tr><td>Lactated Ringer</td><td>Reagent</td><td>Hospira Inc.</td><td>NA</td><td></td></tr><tr><td>Xylocaine-MPF</td><td>Other</td><td>AstraZeneca</td><td>NA</td><td></td></tr><tr><td>Syringe 1mL</td><td>Tool</td><td>BD Biosciences</td><td>309602</td><td></td></tr></tbody></table>

human in-vivo bioassay for the tissue-specific measurement of nociceptive and inflammatory mediators

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

This video begins 24 hours after the induction of the experimental sunburn on the thigh of a patient using a calibrated UV light source. After application of local anesthetic, a microdialysis catheter is inserted between the cuts and subcu and is then pulled through the skin. A perfusion syringe is attached to the proximal end of the catheter, and the activation of the perfusion pump will induce the flow and collection of micro lysate.Following collection samples can be stored at minus 80 degrees Celsius until analysis of nociceptive and inflammatory mediators is ready to be performed. Hi, I'm Martin Angst from the Department of Anesthesia of Stanford University. And hi, I'm Martha Tingle, senior research nurse and manager of the Human Pain Laboratory, founded by Dr.Angst in 1995 here at Stanford University.Today we're gonna show you a procedure for the placement of Microdialysis catheters in experimental inflamed skin of human study participants for the collection of interstitial fluid. We use this technique in our laboratory to study tissue specific release of biochemicals critical for the mediation of pain and inflammation. So let's get started.One model of experimental skin inflammation is the artificial sunburn. A first degree sunburn is induced with aid of a calibrated ultraviolet B source. Accurate delivery of energy is assured with a UV meter.Sunburn is typically induced by applying two to three times the amount of UVB light necessary to cause reding Before collecting the specimens, prepare a staging area with sterile gloves, a sterile needle holder, and one CC lura tip syringes with 23 gauge and 30 gauge needles, you'll also need microdialysis catheters attached at one end to a four centimeter 23 gauge needle samples will be collected in 1.5 milliliter micro cent collection tubes containing four microliters of protease inhibitor solution per 100 microliter sample protease inhibitor solution is made per manufacturer's instruction using protease inhibitor cocktail tablets, prepare a 1%human albumin solution in lactated ringer solution and draw it into the one cc lure Tip syringe, making sure there are no air bubbles remaining in the syringe. Load the syringe with the 1%human albumin enriched ringers lactate solution into the cradle mounted onto a high precision infusion pump. Multiple syringes can be loaded to simultaneously obtain interstitial fluid samples from various sites.Make certain that the bar of the infusion pump driving the syringe is in full contact with the plunger of the syringe. Set the pump rate after confirming that the syringe diameter has been accurately entered into the pump. Commonly used infusion rates range between two to four microliters per minute.Now you are ready to place the catheter prior to catheter placement. The area from which samples are collected must be defined when sampling from an artificially created inflamed area, such as an experimentally induced sunburn. Plan on inserting the catheter at the edge of the sunburn.Clean a small area at opposite edges of the lesion or C site you intend to collect your sample from With alcohol swabs, these cleaned areas will be the intended insertion and exit points for the catheter. Using a one cc syringe with 30 gauge needle inject 1%Lidocaine incu evidenced by laning of the skin on injection, inject just enough lidocaine at these sites to create a very small bleb with a maximum size of two millimeters. Injecting a small amount of lidocaine at the entry and exit points of the microdialysis catheter makes catheter insertion less painful for subjects, but should not affect bioassay results Using aseptic techniques.Insert the microdialysis catheters with the aid of the attached 23 gauge needle over the length of the explored skin area. The depth of insertion is at the interface between the cuts and subcu. Adequate penetration can be verified visually as the needle and the attached catheter are moved forward.The needle should cause a little up folding of the skin as well as some blanching of the skin. No bleeding should occur if bleeding occurs. The needle is located deeper than intended and the depth of insertion has to be adjusted.Once the needle has been pushed through the exit point of the skin, pull gently on the needle to feed the attached catheter through the skin. Gentle handling is critical because the catheters are small diameter and very fragile. Pull the catheter until it is completely threaded through the skin that is the base of the larger plastic tubing.Embedding the distal end of the catheter should touch the skin at the entry point. Again, little to no bleeding should occur indicating that the catheter has been placed correctly. To begin the sample collection, first flush the catheters with 1%albumin enriched ringers lactate solution.This is done by carefully inserting a syringe with a 23 gauge needle into the distal end of the microdialysis catheter and slowly injecting solution. Flush the catheter until you see droplets along the length of the catheter as well as at its tip. After flushing the catheter, attach the distal end of the catheter to the tip of the 23 gauge needle of the syringe that is resting in the rack of the infusion pump.Avoid puncturing the needle through the wall of the catheter, which would cause potential leakage using quarter inch pink tape. Attach the prepared 1.5 milliliter micro cent collection tube at the skin close to the exit side of the catheter. Place the tip of the catheter into the collection tube to allow collection of micro lysate.Start the perfusion pump at the predetermined rate and intermittently check that the delivered and collected volume match up in case the volumes do not match up. Check to see if there is leakage from the skin entry. Site leaks can sometimes be resolved by pulling the part of the catheter that is embedded by the plastic tubing more tightly onto the skin entry site.Alternatively, a catheter may need to be replaced if delivered and collected. Volumes do not match up once sampling is complete. The samples are placed on dry ice until storage at minus 80 degrees Celsius.We have just shown you how to collect interstitial fluid from experimental inflamed skin using microdialysis catheters. We are now ready to analyze obtained samples with immunoassays for biochemical markers of pain and inflammation. When doing this procedure, it's important to handle the catheters very carefully advance the catheters slowly keeping tension off the tubing, and it may be helpful to lubricate the catheters prior to insertion with a little bit of the sterile solution that you've prepared for the infusion phase of the study.So that's it. Thanks for watching and good luck with your experiments.

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Learn microdialysis catheter placement for tissue-specific collection of interstitial fluid from inflamed human skin. This method enables measurement of nociceptive and inflammatory mediators in pain research.

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

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