Microdialysis

Microdialysis is based on diffusion across a semi-permeable membrane due to a concentration gradient. Perfusate, pH and osmotically balanced with plasma, exchanges across a semi-permeable membrane. The dialysate contains molecular contents originating in the interstitium which have entered by passive diffusion. Dual functions are served: medication may be administered to the interstitium in perfusate and interstitial contents may be sampled in dialysate. Relative recovery, the fraction of the biochemical found in the dialysate, is independent of the concentration of the analyte, but depends on flow rate, composition of the perfusate, surface area, physicochemical properties of the microdialysis catheter, and the nature of the sampled site. Given these restrictions and similar application of similar catheters to similar sites at similar pump rates, it is reasonable to assume that differences measured in dialysate reflect differences in biochemical concentrations within the interstitium between patients and controls. Also, it is reasonable to assume similar rates of delivery of perfused drugs. We use microdialysis catheters inserted into the dermal space of the lateral aspect of the calf on although most commonly the non-glabrous skin of the forearm is used. . Catheters contain 10 mm long, 20 kDa membranes with good recovery up to at least 5000 Daltons. A 25 gauge needle is inserted into the dermal space and exits the skin approximately 15 mm distant. The microdialysis catheter is inserted into the lumen of the needle, and withdrawn so that the dialysis membrane is 10 mm from the point of insertion. Once the membrane is placed, the needle is withdrawn. Multiple catheters are typically used but must be spaced at least 6 cm apart throughout all procedures and are taped in place. This is to avoid spurious effects of the axon reflex. Perfusion rates are usually at 2μl/min in studies. Thus, rather small quantities of perfusate are delivered and dialysate are sampled. Chemicals are dissolved in Ringer solution. Cutaneous flow is monitored by LDF. Before measurements are performed, hyperemia related to trauma will need to abate as indicated by the return of laser Doppler flow rates to approximate baseline levels. This takes approximately at least 60 minutes and may be much longer depending on the subject. The flow measured is affected by the optical properties of the skin and other factors. It is usually reported in arbitrary perfusion units. Most often investigators express results as a percent of maximum conductance of the skin frequently obtained by using high dose sodium nitroprusside at the end of experiments.  Up ]

 

 

 The figure shows the process of placing intradermal microdialysis catheters and the placement of integrating laser-Doppler probes over the central site where the microdialysis membrane is located. Some of the probes include a thermal collar used to heat the surrounding tissue.
 

 

In research on POTS we have made use of two microdialysis dependent assays for nitric oxide (NO): the first shown in the panels above is a method using gradual heating of an area resulting in a biphasic response shown initially by Kellogg and Minson. There is  an initial peak which can be suppressed by local anesthetics and is axon related. Following a nadir there is a plateau phase that occurs about 30 minutes after heat is initiated. As shown in the right hand panel, the plateau phase is dependent on nitric oxide and more specifically on nNOS.
The second NO assay method is a dose-response to acetylcholine delivered by intradermal microdialysis. As shown in the adjoining panel this is highly NO sensitive, but relatively independent of nNOS or iNOS.  
 
 

 


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