Cytokine and Chemokine Recovery Is Increased by Colloid Perfusates during Dermal Microdialysis.

Sven Quist,Harald Gollnick,Claudia Kirbs,Charlotte Kloft

doi:10.3390/ma11050682

Abstract

Cytokines and chemokines play important roles in cell signalling, and microdialysis is a promising tool for monitoring these inflammation markers ex vivo. Therefore, the collecting of these mediators at the highest concentrations possible is crucial. Depending on the size of the mediator of interest, the collection of these high molecular mass molecules has thus far been difficult due to their low recovery, even when using high cut-off (100 kDa) microdialysis membranes. This study aimed to optimize the recovery of various cytokines and chemokines by validating the use of different perfusates in cutaneous microdialysis, and comparing intravenous (i.v.) colloids, crystalloids, and a lipid emulsion formulations that are approved for i.v. applications. Methods: In vitro and in vivo recovery experiments using six recombinant cytokines varying in molecular size (interleukin-2 (15 kDa), interleukin-6 (20.5 kDa), interleukin-8 (8 kDa), interleukin-12p70 (70 kDa), TNF-α (17.5 kDa), and vascular endothelial growth factor (VEGF) (38 kDa)) were performed in the presence of different perfusates for i.v. applications: Ringer’s lactate, dextran 60 kDa, hydroxyethyl starch 70 kDa, and hydroxyethyl starch 200 kDa solutions as well as a lipid emulsion formulation. Recovery was determined through (i) microdialysis of cytokines and chemokines in Ringer’s lactate solution or human serum in vitro, and (ii) retrodialysis of excised porcine and human skin cadavers in vitro and porcine skin in vivo. Furthermore, we used skin trauma (catheter insertion) and Ultraviolet B irradiation of 3 × 3 cm2 skin areas to sample cytokines and chemokines in vivo and compared the amounts that were obtained using crystalloid and colloid perfusates. All the cytokines and chemokines within the dialysates were quantified through a flow cytometry-based bead array assay. Results: Overall, recovery was strongly increased by the colloids, particularly hydroxyethyl starch 70 kDa, in vitro, ex vivo, and in vivo. When compared with the recovery achieved using Ringer’s lactate, this increase was most effective for proteins ranging from 8 to 20.5 kDa. Hydroxyethyl starch 70 kDa significantly increased the recovery of interleukin (IL)-8 in human serum in vitro when compared with Ringer’s lactate. More cytokines and chemokines were recovered using colloids compared with crystalloids. However, the increase in recovery values was lower for IL-12p70 and VEGF. Conclusions: Regarding the dialysate volumes and final dialysate concentrations, colloid perfusates are overall superior to crystalloid perfusates, such as Ringer’s lactate, when sampling cytokines and chemokines, resulting in higher recoveries. However, the sampling of high-molecular-mass cytokines during microdialysis remains challenging, and experimental in vitro data are not completely comparable with data obtained ex vivo or in vivo.

Highlights

Dermal microdialysis (DM) is a method currently used to monitor tissue chemistry in vivo, including tissue physiology, pathophysiology, drug efficacy, and drug tissue concentrations, and is feasibly applicable to almost every human organ, including skin [1,2,3,4,5,6]
Sampling macromolecules, including high-molecular-mass proteins, such as cytokines and chemokines, via microdialysis has gained increasing interest, as it is the only method that allows for the in vivo detection of intercellular signalling molecules without destroying the tissue of interest [17,24]
Low recovery of high-molecular-mass proteins and sampling intervals of up to 8 h have reportedly yielded a sufficient amount of analytes, even when using high cut-off microdialysis membranes of 100 kDa [9,11], which is very unlike the actual in vivo environment

Summary

Introduction

Dermal microdialysis (DM) is a method currently used to monitor tissue chemistry in vivo, including tissue physiology, pathophysiology, drug efficacy, and drug tissue concentrations, and is feasibly applicable to almost every human organ, including skin [1,2,3,4,5,6]. This catheter functions as an artificial vessel and is used to perfuse the tissue with a tissue-compatible sterile buffer at a low flow rate by means of a microdialysis pump (Figure 1A) [1,2,7,8]. Conventional sampling of high-molecular-mass proteins (>10 kDa) remains challenging due to their low recovery, even when higher cut-off membranes of 100 kDa are used [15,16,17,18]. Low recovery of macromolecules, such as high-molecular-mass cytokines and chemokines, is influenced by different levels of colloid osmotic pressure on both sides of a semipermeable membrane. In cases of crystalloid perfusates, such as 0.9% NaCl or Ringer’s lactate solutions, the colloid osmotic pressure outside the microdialysis membrane in the tissue of interest prevents the diffusion of macromolecules into the catheter lumen

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Conclusion