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

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.