Fluorescent imaging analysis of dextran leakage across large sized pores brain microdialysis probes

Microdialysis (MD) is a minimally invasive diffusion based method for a time continuous sampling of drugs and endogenous compounds present in extracellular fluid and for the drug delivery directly at the site of interest. Large molecular weight cut off (CO) MD is a new area of application of MD focused on sampling large biomolecules e.g. cytokines and other proteins. Large CO probes have a high risk of the convective transmembrane transport that would lead to a not negligible perturbation of the external microenvironment. One way to inhibit the convective flow is to adjust the transmembrane pressure by adding dextrans as osmotic agents to the perfusion fluid. It is crucial that when dextrans are used they do not leak out into the tissue since it would alter again the sampling microenvironment and might cause physiologial responses of the organism. The declared by the constructor CO seems from the previous studies to be not reliable to predict the behavior of different type molecules. Therefore, the behavior of dextrans is important to study. This project performed a comparison of leakage of different molecular weight dextrans (40 kDa, 70 kDa, 250 kDa, 500 kDa) through the large sized pores membrane (100 kDa CO) of the brain MD probe. This was done by fluorescent imaging analysis, in the attempt to validate the fluorescence microscopy (FM) as a tool for qualitative and quantitative study of the MD. A MD probe holder device was designed and manufactured with micromachining techniques, mainly up of polidymetilsyloxane and glass. Qualitative image analysis showed MD process and MD probe details. A method for quantitative imaging analysis was developed. The method consisted in use of Matlab code to perform the sum of pixel brightness values over the normalized image of dextrans leakage pattern. The measurement resulted to be repeatable. Probes of the same type resulted to have different leaking properties. Although the MWCO of the membrane was 100 kDa some of 250 kDa dextrans leaked out anyway. There was no leakage of 500 kDa dextrans. Concentration loss during the perfusion was also assessed from pictures of inflow and outflow tubes and was consistent with leakage data.