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MICRODIALYSIS

Chapter 1

INTRODUCTION

Microdialysis is a minimally-invasive sampling technique that is used for continuous

measurement of free, unbound analyte concentrations in the extracellular fluid of virtually

any tissue. Analytes may include endogenous molecules to assess their biochemical

functions in the body, or exogenous compounds to determine their distribution within the

 body. The microdialysis technique requires the insertion of a small microdialysis catheter 

(also referred to as microdialysis probe into the tissue of interest. The microdialysis probe is

designed to mimic a blood capillary and consists of a shaft with a semipermeable hollow

fiber membrane at its tip, which is connected to inlet and outlet tubing. The probe iscontinuously perfused with an aqueous solution (perfusate that closely resembles the (ionic

composition of the surrounding tissue fluid at a low flow rate of approximately !."-#$%&min.

[1]'nce inserted into the tissue or (bodyfluid of interest, small solutes can cross the

semipermeable membrane by passive diffusion. The direction of the analyte flow is

determined by the respective concentration gradient and allows the usage of microdialysis

 probes as sampling as well as delivery tools.[1] The solution leaving the probe (dialysate is

collected at certain time intervals for analysis.

 

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Chapter 2

R!"TD T#OR$

The microdialysis principle was first employed in the early ")!s, when push-pull

canulas *+ and dialysis sacs * were implanted into animal tissues, especially into rodent

 brains, to directly study the tissues biochemistry.*" /hile these techniques had a number of 

experimental drawbac0s, such as the number of samples per animal or no&limited time

resolution, the invention of continuously perfused dialytrodes in "1+ helped to overcome

some of these limitations.*23urther improvement of the dialytrode concept resulted in the

invention of the 4hollow fiber4, a tubular semipermeable membrane with a diameter of 5+!!-

!!$m, in "12.*# Todays most prevalent shape, the needle probe, consists of a shaft with a

hollow fiber at its tip and can be inserted by means of a guide cannula into the brain and other 

tissues. 

2%1 &ICRODI"!$'I' (RO)'

3ig +." 6chematic illustration of a micro dialysis probe

There are a variety of probes with different membrane and shaft length combinations

available. The molecular weight cutoff of commercially available micro dialysis probes

covers a wide range of approximately )-"!!07, but also "M7 is available. /hile water-

soluble compounds generally diffuse freely across the microdialysis membrane, the situation

is not as clear for highly lipophilic analytes, where both successful (e.g. corticosteroids and

unsuccessful microdialysis experiments (e.g. estradiol, fusidic acid have been reported.

*) 8owever, the recovery of water-soluble compounds usually decreases rapidly if the

molecular weight of the analyte exceeds +#9 of the membrane:s molecular weight cutoff.

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MICRODIALYSIS

2%2 RCO*R$ "ND C"!I)R"TION &T#OD'

7ue to the constant perfusion of the microdialysis probe with fresh perfusate, a total

equilibrium cannot be established.*"

 This results in dialysate concentrations that are lower than those measured at the distant sampling site. ;n order to correlate concentrations

measured in the dialysate with those present at the distant sampling site, a calibration factor 

(recovery is needed. The recovery can be determined at steady-state using the constant rate

of analyte exchange across the microdialysis membrane. The rate at which an analyte is

exchanged across the semipermeable membrane is generally expressed as the analyte:s

extraction efficiency. The extraction efficiency is defined as the ratio between the loss&gain of 

analyte during its passage through the probe (<in-<out and the difference in concentration between perfusate and distant sampling site (<in-<sample. ;n theory, the extraction

efficiency of a microdialysis probe can be determined by= " changing the drug

concentrations while 0eeping the flow rate constant or + changing the flow rate while

0eeping the respective drug concentrations constant. At steady-state, the same extraction

efficiency value is obtained, no matter if the analyte is enriched or depleted in the perfusate.

*" Microdialysis probes can consequently be calibrated by either measuring the loss of analyte

using drug-containing perfusate or the gain of analyte using drug-containing sample

solutions. To date, the most frequently used calibration methods are the low-flow-rate

method, the no-net-flux method,*1 the dynamic (extended no-net-flux method,*> and the

retrodialysis method.* The proper selection of an appropriate calibration method is critically

important for the success of a microdialysis experiment. 6upportive in vitro experiments prior 

to the use in animals or humans are therefore recommended. *" ;n addition, the recovery

determined in vitro may differ from the recovery in humans. ;ts actual value therefore needs

to be determined in every in vivo experiment.*)

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MICRODIALYSIS

2%2%1 !o+,flo+,rate method

The low-flow-rate method is based on the fact that the extraction efficiency is

dependent on the flow-rate. At high flow-rates, the amount of drug diffusing from the

sampling site into the dialysate per unit time is smaller (low extraction efficiency than at

lower flow-rates (high extraction efficiency. At a flow-rate of ?ero, a total equilibrium

 between these two sites is established (<out @ <sample. This concept is applied for the

(low-flow-rate method, where the probe is perfused with blan0 perfusate at different flow-

rates. <oncentration at the sampling site can be determined by plotting the extraction ratios

against the corresponding flow-rates and extrapolating to ?ero-flow. The low-flow-rate

method is limited by the fact that calibration times may be rather long before a sufficient

sample volume has been collected.

2%2%2 No,net,flux,method

7uring calibration with the no-net-flux-method, the microdialysis probe is perfused

with at least four different concentrations of the analyte of interest (<in and steady-state

concentrations of the analyte leaving the probe are measured in the dialysate (<out. *1 The

recovery for this method can be determined by plotting <out-<in over <in and computing the

slope of the regression line. ;f analyte concentrations in the perfusate are equal to

concentrations at the sampling site, no-net flux occurs. espective concentrations at the no-

net-flux point are represented by the x-intercept of the regression line. The strength of this

method is that, at steady-state, no assumptions about the behaviour of the compound in the

vicinity of the probe have to be made, since equilibrium exists at a specific time and place.

*)8owever, under transient conditions (e.g. after drug challenge, the probe recovery may be

altered resulting in biased estimates of the concentrations at the sampling site. To overcome

this limitation, several approaches have been developed that are also applicable under non-

steady-state conditions. 'ne of these approaches is the dynamic no-net-flux method .

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MICRODIALYSIS

2%2%- D.namic no,net,flux method

/hile a single subBect&animal is perfused with multiple concentrations during the no-

net-flux method, multiple subBects are perfused with a single concentration during the

dynamic no-net-flux (7CC3 method.*> 7ata from the different subBects&animals is then

combined at each time point for regression analysis allowing determination of the recovery

over time. The design of the 7CC3 calibration method has proven very useful for studies that

evaluate the response of endogenous compounds, such as neurotransmitters, to drug

challenge.

2%2%/ Retrodial.sis

7uring retro dialysis, the micro dialysis probe is perfused with an analyte-containing

solution and the disappearance of drug from the probe is monitored. The recovery for this

method can be computed as the ratio of drug lost during passage (<in-<out and drug

entering the microdialysis probe (<in. ;n principle, retrodialysis can be performed using

either the analyte itself (retrodialysis by drug or a reference compound (retrodialysis by

calibrator that closely resembles both the physiochemical and the biological properties of the

analyte.* 7espite the fact that retrodialysis by drug cannot be used for endogenous

compounds as it requires absence of analyte from the sampling site, this calibration method ismost commonly used for exogenous compounds in clinical settings.

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MICRODIALYSIS

Chapter -

(RINCI(! O0 &ICRODI"!$'I'

Microdialysis is a minimally invasive technique allowing in vivo sampling of 

molecules transported into, or generated within the extracellular space of principally any

tissue or organ in the body and also for sampling the body fluids such as blood or <63.

Microdialysis in combination with a suitable detection technique allows monitoring of time-

dependent changes in local tissue chemistry, for example neurotransmitter release and

reupta0e, drug delivery or energy metabolism in a particular brain area. The unsurpassed

feature of in vivo microdialysis is its capability to provide information on basal, non-

stimulated levels of extracellular neurotransmitters, as well as, on pharmacologically or  physiologically stimulated release. This offers a unique opportunity to examine the role of 

various receptor subtypes in tonic and phasic regulation of neurotransmitter release and

metabolism in neuroanatomically relevant brain structures.

Microdialysis technique provides the most comprehensive information on dynamic

changes of molecules involved in intercellular communication and metabolism at an

integrative (whole body level, preserving the overall physiological and behavioral functions.

6everal independent techniques have demonstrated that the molecular movement within the

extracellular space is driven predominantly by diffusion. ;n an analogous way, the driving

force of microdialysis sampling is the diffusion of molecules across the concentration

gradients existing between the two compartments separated by the membrane= the

tissue&extracellular space compartment and the perfusion fluid inside the probe as the second

compartment. Thus, the molecules can move in both directions, which allows simultaneous

recovery of endogenous compounds released into the brain microenvironment and at the

same time, drugs can be delivered locally through the probe into the sampled

area.Microdialysis can be used for monitoring the 0inetics of drug distribution and clearance

in different organs, which for the brain studies is of particular interest since it allows

evaluating the ability of drugs to penetrate through the blood-brain barrier.

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MICRODIALYSIS

The principle of microdialysis sampling and the origin of molecules released into the brain

microenvironment. The molecular movement of substances present in the extracellular fluid

is driven predominantly by diffusion. 7epending on the concentration gradients across the

membrane of the microdialysis probe, the molecules can move in both directions, which

allows simultaneous recovery of endogenous compounds and at the same time, drugs can be

added to the perfusion medium and delivered locally into the same brain structure

The most interesting and thoroughly studied molecules are (depicted by numbers= ",+, -

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MICRODIALYSIS

neurotransmitters, neuromodulators and neuropeptidesD ,2 - the neuron-glia interactions,

glutamate and EAFA, large molecules such as interleu0ins and trophic factorsD # - second

messengers cAMG, cEMG or arachidonic acid metabolitesD ) - molecules transported from

 blood capillaries - glucose, nutrients, drugsD 1 - neuro-vascular communication - C'D > -

molecules transported from or into the <63.

Adopted from= Hehr I, Joshita0e T (+!!) Monitoring brain chemical signals by

microdialysis. ;n= Kncyclopedia of 6ensors (Kds <A Erimes, K< 7ic0ey and ML Gish0o

American 6cientific Gublishers, 6A, pp +>1-"+.

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MICRODIALYSIS

C#"(TR

"D*"NT"'

". To date, microdialysis is the only sampling technique that can continuously monitor 

drug or metabolite concentrations in the extracellular fluid of virtually any tissue.

7epending on the exact application, analyte concentrations can be monitored over several hours, days, or even wee0s. 3ree, unbound extracellular tissue concentrations

are in many cases of particular interest as they resemble pharmacologically active

concentrations at or close to the site of action. <ombination of microdialysis with

modern imaging techniques, such positron emission tomography, further allow for 

determination of intracellular concentrations.

+. ;nsertion of the probe in a precise location of the selected tissue further allows for 

evaluation of extracellular concentration gradients due to transporter activity or other 

factors, such as perfusion differences. ;t has, therefore, been suggested as the most

appropriate technique to be used for tissue distribution studies.

. Kxchange of analyte across the semipermeable membrane and constant replacement

of the sampling fluid with fresh perfusate prevents drainage of fluid from the

sampling site, which allows sampling without fluid loss. Microdialysis can

consequently be used without disturbing the tissue conditions by local fluid loss or 

 pressure artifacts, which can occur when using other techniques, such as

microinBection or push-pull perfusion.

2. The semipermeable membrane prevents cells, cellular debris, and proteins from

entering into the dialysate. 7ue to the lac0 of protein in the dialysate, a sample clean-

up prior to analysis is not needed and en?ymatic degradation is not a concern.

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MICRODIALYSIS

Chapter 3

!I&IT"TION'

". 7espite scientific advances in ma0ing microdialysis probes smaller and more efficient,

the invasive nature of this technique still poses some practical and ethical limitations. 3or 

example, it has been shown that implantation of a microdialysis probe can alter 

tissue morphology resulting in disturbed microcirculation, rate of metabolism or integrity

of physiological barriers, such as the bloodNbrain barrier .*" /hile acute reactions to

 probe insertion, such as implantation traumas, require sufficient recovery time, additional

factors, such as necrosis, inflammatory responses,*"" or wound healing processes have to

 be ta0en into consideration for long-term sampling as they may influence the

experimental outcome. 3rom a practical perspective, it has been suggested to perform

microdialysis experiments within an optimal time window, usually +2N2> hours after 

 probe insertion.*"2*"#

+. Microdialysis has a relatively low temporal and spatial resolution compared to, for 

example, electrochemical biosensors. /hile the temporal resolution is determined by the

length of the sampling intervals (usually a few minutes, the spatial resolution is

determined by the dimensions of the probe. The probe si?e can vary between different

areas of application and covers a range of a few millimeters (intracerebral application up

to a few centimeters (subcutaneous application in length and a few hundred micrometers

in diameter.

. Application of the microdialysis technique is often limited by the determination of the

 probe:s recovery, especially for in vivo experiments. 7etermination of the recovery may

 be time-consuming and may require additional subBects or pilot experiments. The

recovery is largely dependent on the flow rate= the lower the flow rate, the higher the

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MICRODIALYSIS

recovery. 8owever, in practice the flow rate cannot be decreased too much since either 

the sample volume obtained for analysis will be insufficient or the temporal resolution of 

the experiment will be lost. ;t is therefore important to optimi?e the relationship between

flow rate and the sensitivity of the analytical assay. The situation may be more complex

for lipophilic compounds as they can stic0 to the tubing or other probe components,

resulting in a low or no analyte recovery.

Chapter 4

CONC!UTION

  The technique of microdialysis enables sampling and collecting of small-

molecular-weight substances from the interstitial space. ;t is a widely used method in

neuroscience and is one of the few techniques available that permits quantification of 

neurotransmitters, peptides, and hormones in the behaving animal. More recently, it has been

used in tissue preparations for quantification of neurotransmitter release. This unit provides a

 brief review of the history of microdialysis and its general application in the neurosciences.

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R0RNC'

". O Iump up to=a b c d  e  f   g  <haurasia <.6., MPller M., Fashaw K.7., Fenfeldt K.,

Folinder I., Fulloc0 ., Fungay G.M., 7e%ange K.<., 7erendorf 8., Klmquist /.3.,

8ammarlund-denaes M., Iou0hadar <., Hellogg 7.%. Ir., %unte <.K. Cordstrom

<.8., ollema 8., 6awchuc0 .I. <heung F./., 6hah L.G., 6tahle %., ngerstedt .,

/elty 7.3. and Jeo 8. (+!!1. 4AAG6-37A /or0shop /hite Gaper= Microdialysis

Grinciples, Application and egulatory Gerspectives4. Pharm Res. 2/ (#= "!"2N 

+#.doi="!."!!1&s""!#-!!)-+!)-?. GM;7 "12#>)>#.

+. 5ump up6 Eaddum I.8. (")". 4Gush-pull cannulae4. J. Physiol. 1= "N+.

. 5ump up6 Fito %., 7avson 8., %evin K.M., Murray M. and 6nider C. (")).

4The concentration of free amino acids and other electrolytes in cerebrospinal fluid,

in vivo dialysate of brain and blood plasma of the dog4.  J. Neurochem. 1- (""=

"!#1N"!)1.doi="!.""""&B."21"-2"#.")).tb!2+)#.x . GM;7 #+2)#1.

2. O Iump up to=a b 7elgado I.M.. and 7e3eudis 3.L., oth .8., yugo 7.H.

and Mitru0a F.M. ("1+. 47ialytrode for long-term intracerebral perfusion in awa0e

mon0eys4. Arch. Int. Pharmacodyn. Ther. 178 ("= 1N+". GM;7 2)+)21>.

#. 5ump up6 ngerstedt . and Gycoc0 <. ("12. 43unctional correlates of 

dopamine neurotransmission4. Bull. Schweiz. Akad. Med. Wiss. -9 ("N= 22N 

##. GM;7 21")#).

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