microdialysis—theory and application

Progress in Neurobiology Vol. 35, pp. 195 to 215, 1990 0301-0082/90/$0.00 + 0.50 Printed in Great Britain. All rights reserved © 1990 Pergamon Press pie

MICRODIALYSIS--THEORY AND APPLICATION

HELENE BENVE~Sa'E* and PETER CHRISTIAN HOTI~mIERt

*Neurology Research Laboratory, Department of Medicine (Neurology), Duke University Medical Center, Durham, NC 27710, U.S.A.

~fDepartment of Anesthesiology, Duke University Medical Center, Durham, NC 27710, U.S.A.

(Received 26 February 1990)

C O N T E N T S

1. Introduction 195 2. Principle of microdiaiysis 196 3. Practical aspects 196

3.1. Implantation of the microdialysis probe in brain tissue 196 3.2. Apphcation in other tissues 197 3.3. Perfusion fluids 197

4. Measurements with microdialysis 199 4.1. Concentration measurements based on/n vitro calibration 200

5. Factors affecting substance collection with microdialysis 200 5.1. Perfusion flow rate and time 200 5.2. Flux 201 5.3. Temperatures and changes in outer substance concentration 201 5.4. Dialysis membrane area and composition 202 5.5. Diffusion coefficient 202

6. Effect of microdialysis on the brain microenvironment 203 6.1. Brain compartmentation 203 6.2. Tissue reactions 203 6.3. Local cerebral blood flow and 2-deoxyglucose phosphorylation 203 6.4. Use of tetrodotoxin and calcium depletion for estimating functional damage after implantation 204 6.5. The possible influence of the disturbed brain mieroenvironment on results obtained by mierodialysis 204

7. Solutions to the problem of determining brain interstitial concentrations with microdialysis 204 7.1. Methods without time resolution 204 7.2. Methods with time resolution 205

8. Transformation of glutamate and dopamine dialysate concentrations into brain interstitial concentrations 207 8.1. Normal brain tissue 207

8. I. 1. Glutamate 207 8.1.2. Dopamine 207

8.2. Interpretation of calculated interstitial glutamate and dopamine concentrations obtained by microdialysis 207 8.3. Glutamate concentrations in isehemic brain tissue 208

9. Conclusion 208 References 209

1. INTRODUCTION

Accurate and continuous in vivo measurements of brain interstitial substance concentrations provide a major technical challenge. Among the many innovations that have appeared over the last decades i.e. cortical cup (reviewed by Moroni and Pepeu, 1984), push-pull cannula (reviewed by Phillippu, 1984), ion-selective mieroelectrodes (Thomas, 1978; Zeuthen, 1981) and carbon fiber microelectrodes (Marsden et al., 1984), microdialysis is in principle the only technique which can collect virtually any substance from remote brain regions with a limited amount of tissue trauma. Described as early as 1966 by Bito et aL, the method has undergone several technical improvements (Delgado et aL, 1972; Ungerstedt et aL, 1982; Hamberger et al., 1983;

Ungerstedt, 1984) and is rapidly becoming a very popular bioanalytical sampling tool in brain research. However, despite the initial excitement surrounding the concept of mierodialysis and its seemingly limit- less applications, this technique like any other is obviously not without problems. Some of these were broadly discussed in a previous report that sum- marized the many different brain substance concentration measurements that had been performed so far (Benveniste, 1989). In reviewing all these data it is clear that certain uniform guidelines and standards are necessary if mierodialysis is to become truly useful in the future. We will attempt to deal with these issues in the following by including a description of some practical and technical aspects, a brief overview of the many applications and an evaluation of the tissue disturbances caused by the implantation procedure.

195

196 H. BENVENISTE and P. C. HOTTEMEIER

The main focus will be on brain microdialysis and solutions to the problem of calculating interstitial substance concentrations from the data obtained by microdialysis. This effort we believe would be of dubious value unless it was put into perspective with some practical examples. Therefore part of this discussion is centered around the measurement of two neurotransmitters, dopamine and glutamate, in normal and ischemic brain.

2. PRINCIPLE OF MICRODIALYSIS

The microdialysis technique uses the dialysis principle and consists of a membrane permeable to water and small solutes which is continuously flushed on one side with a solution devoid of the substances of interest, whereas the other side faces the interstitial space (this obviously only applies for substance collection; if a substance is instead to be delivered, it must be present in the perfusion fluid in a higher concentration than that of the outer medium). A concentration gradient is created causing diffusion of substances from the interstitial space into the dialysis membrane (and vice versa) as illustrated in Fig. 1. The continuous flow through the membrane carries substances to the sampling site for further analysis. A dialysis membrane connected with in- and outlet tubes, allowing fluid to enter and leave the dialysis membrane, respectively, is usually referred to as a 'microdialysis probe'. There are two main types of microdialysis probes: (a) dialysis membranes, formed

brain Interst i t ial fluid

Serial

Parallel

I

Loop

_ _ I ~

Side by slde Concentric

FIG. 2. Designs. Radial sections of a serial microdialysis probe and three different mierodialysis probes with in- and outlet tubes positioned in parallel. The outer diameter of the dialysis membrane is usually 300~00 #M. The length of the

membrane is adjusted to the brain regions studied.

as cylinders, connected with in- and outlet tubes in a serial arrangement, and (b) those with in- and outlet tubes positioned in parallel as illustrated in Fig. 2.

0

0 0

0

0

0 0

0 0 0

o ®1 o

o t = O

0

0

d~alysla membrane

• O o ~ ° D

,0 ~) • 0

"¢" o. ( I t O

' O • • t 0

L 0 10 • t~O

O

FIG. !. The dialysis principle. The basic principle is the positioning of a membrane that allows free diffusion of water and small solutes between the solution of interest (brain interstitial space) and a solution lacking the substances concerned. Because the perfusion fluid is constantly renewed, a concentration gradient is created across the membrane. This forms the basis for diffusion of substances from brain interstitial space into the dialysis membrane. For

further details see Section 2.

3. PRACTICAL ASPECTS

3.1 . IMPLANTATION OF THE MICRODIALYSIS

PROBE XN BRAIN T~ssuE

The brain is especially well-suited for microdialysis because the blood-brain barrier prevents a rapid exchange of most hydrophilic substances between the brain interstitial space and the vascular compartment (Fig. 3). When implanting a microdialysis probe into the brain the animal is anesthetized and positioned in a stereotaxic frame. Having assessed the size of the region of interest it is important to coat and seal off those areas of the microdialysis membrane that may come into contact with adjacent brain regions. The positioning of the probe requires--depending on the probe design--one or two craniectomies on either side of the skull over the brain region of interest. The dura is incised prior to insertion because the dialysis membrane is otherwise easily damaged. During implantation (by means of a micromanipulator attached to the stereotaxic apparatus) the microdialysis probe is usually perfused. This is essential for some dialysis membrane types that do not tolerate drying out and is recommended in general as a means of checking membrane integrity. The microdialysis probe is usually perfused 30 min-2 hr before experiments are carried out so as to obtain 'stable' baseline values (for further details see Sections 5 and 6). For short- or long-term measurements in the awake freely moving

MICRODIALYSIS---THEORY AND APPLICATION 197

interstitial ~ l 1 compartment

A O • s

compartment

• • Oo

FIG. 3. Brain compartmentation. The brain comprises three different water spaces: The intracellular, the interstitial and the vascular space. The microdialysis membrane has direct access to the interstitial fluid compartment. For further

details see Sections 3 and 5.

animal the microdialysis probe is either fixed to the skull after implantation or implanted via a guide which is fixed to the skull (for further details see Lehmann et al., 1983; Delgado et al., 1984; Imperato and Di Chiara, 1984; Ungerstedt, 1984; Korf and Venema, 1985; Hernandez et al., 1986; Collin and Ungerstedt, 1988).

3.2. APPLICATION IN OTHER TISSUES Although the microdialysis principle was originally

intended for use in brain (Bito et al., 1966; Delgado et al., 1972; Ungerstedt and Pycock, 1974) and later used extensively in this organ (cf. Table l) the range of applications appear to be expanding. A variety of other organs and tissues have been studied with microdialysis (cf. Table 2). The true advantage of this remains to be seen because these organs unlike the brain have no tight barriers between the blood stream and the interstitial space. On the other hand microdialysis should be able to provide much needed information on regional metabolic events that cannot be detected by simple downstream blood sampling. Except for microdialysis in eye (Ben-Nun eta[ . , 1988) and heart muscle (Hamberger, 1989), the implantation procedure in other tissues seems to be straight- forward (Bito et al., 1966; Delgado et al., 1972; Meirieu et al., 1986; Pilowsky et al., 1986; Brodin et al., 1987; L6nnroth et al., 1987; Arner et al., 1988; Hurd et al., 1988; Speciale et al., 1988; Lchmann, 1989; Panter et al., 1990). Ben-Nun et al. (1988) have described in detail the surgical procedures for implantation of a microdialysis probe into the vitreous cavity of the eye. Microdialysis in heart muscle is still at a developmental stage (Hamberger, 1989) and likely very problematic due to difficulties in anchoring the membrane to the moving muscle. Microdialysis in blood appears particularly useful, because it represents a way of continuous sampling without drawing any blood (Arner et al., 1988; Hurd et al., 1988; Speciale et al., 1988). This is obviously a great advantage in small animals with limited blood volumes.

3.3. PERFUSION FLUIDS

Table 3 shows the variety of perfusion fluids that are used for microdialysis. It can be seen that these

vary widely with respect to composition and p H - - they are 'physiological' (as frequently quoted) in the sense that they are isosmotic with plasma and this is important. For instance perfusion with anisosmotic media changes the dialysate concentration of taurine collected from brain (Soils et al., 1988; Wade et al., 1988; Lehmann, 1989) and muscle (Lehmann, 1989), respectively. Another problem may arise when the investigator wants to measure a substance that is already contained in the perfusion medium. This necessitates a substitution of the substance of interest in accordance with the principles of diffusion (cf. Section 2) while at the same time maintaining the osmolarity of the perfusion fluid. The result may be a change of the initial control conditions in the surrounding tissue, e.g. substitution of choline for sodium has recently been shown to evoke a selective increase of interstitial taurine (Lehmann and Sandberg, 1990). On the other hand, isosmolar re- placement of NaCl by sucrose does not seem to affect interstitial amino acid levels (Solis et al., 1988).

A physiological fluid is defined as one which is isotonic with respect to blood serum, i.e. of the same osmotic concentration (osmolarity of blood serum = 300 mmol) and also composition. Perfusion fluids that are identical to plasma (for example Ringers solutions) have ion concentrations that differ from those of the brain interstitial space. Normally, the brain possesses homeostatic mechanisms that maintain constant ion concentration in the interstitial fluid and cerebrospinal fluid (CSF) despite variations in plasma composition (Bradbury, 1979). The homeostatic mechanisms are especially efficient for ions such as K +, Ca 2+, and Mg 2+. They operate in conjunction with a low ion permeability in brain capillaries and active ion-transport mechanisms in the choroid plexus and brain endothelial cells (Bradbury, 1979; Hansen, 1985). The interstitial fluid and the CSF are thus protected against changes in plasma ion composition. The dialysis membrane on the other hand has direct access to the interstitial fluid compartment (Fig. 3) and if the latter is perfused with Ca2+-free fluids, interstitial Ca 2+ is drained in a wide area around the dialysis membrane (Fig. 4) (Benveniste et al., 1989). Under such circumstances both active regulatory mechanisms and mass transport by diffusion in the interstitial space are apparently not operating sufficiently fast to permit normal interstitial ion-homeostasis. Interstitial calcium depletion causes dialysate concentrations of various transmitters to decrease (Imperato and Di Chiara, 1984; Westerink and De Vries, 1988). Likewise, if the perfusion fluid contains calcium in a higher concentration than that of the interstitial space (i.e. Ringer's solutions) basal dialysate concentrations of dopamine increase by 70% and the response of dopamine to ~:methyl- tyrosine (an inhibitor of tyrosine hydroxylase) changes when compared to a perfusion fluid containing a Ca 2+ concentration identical to that of the brain interstitial space (Moghaddam and Bunney, 1989). Consequently it appears to be important to keep the ion composition of the perfusion fluid identical with that of the brain interstitial fluid and not with plasma [unless of course a local lowering of interstitial ion concentrations is intended (Imperato and Di Chiara, 1984; Soils et al., 1986; Westerink and De Vries, 1988;

198 H. BENVENISTE and P. C. Hf]TTEMEIER

TABLE 1. APPLICATIONS IN BRAIN AND SPINAL CORD

Ischemia-Hypoxla-Anoxia

Benveniste et al., 1984; Hagberg et al., 1984; Hagberg et aL, 1985; Drejer et al., 1985; Hagberg et al., 1986; Lindefors et al., 1986; Phebus et al., 1986; Van Wylen et al., 1986; Hagberg et al., 1987a; Hagberg et al., 1987b; Benveniste and Diemer, 1988a; Globus et al., 1988a; Globus et al., 1988b; Kendrick and Leng, 1988; K o r f e t al., 1988; Slivka et al., 1988; Vulto et al., 1988; Benveniste et aL, 1989b; Busto et al., 1989; Globus et al., 1989; Hillered et al., 1989; Ikeda et al., 1989; Phebus and Clemens, 1989; Shimada et al., 1989.

Hypoglycemia

Tossman et al., 1985; Sandberg et al., 1986; Butcher et al., 1987a; Butcher et al., 1987b.

Seizures-Hyperexcitation-Kainic acid Lehmann et al., 1983; Lehmann et al., 1984; Jacobsen and Hamberger, 1985; Lehmann et al., 1985a; Vezzani et al., 1985; Lazarewicz et al., 1986a; Lehmann et al., 1986; Pilowsky et al., 1986; Butcher et al., 1987c; Vezzani et al., 1988; Young et al., 1988.

Injury Faden et al., 1989; Panter et al., 1990.

Hepatic encephalopathy Tossman et al., 1983; Hamberger and Nystr6m, 1984; Tossman et al., 1987.

Behavior Ungerstedt et al., 1982; Sharp et al., 1987b; Kendrick et al., 1986; Kendrick et al., 1988a; Kendrick, 1988b; Abercrombie et al., 1989.

Dopamine Ungerstedt and Pycock, 1974; Hernand6z et al., 1983; Zetterstr6m et al., 1983; Blakely et al., 1984; Clemens and Phebus, 1984; Imperato and Di Chiara, 1984; Sharp et aL, 1984; Kite et al., 1986; Sharp et aL, 1986a; Sharp et al., 1986b; Bettini et al., 1987; Wood et al., 1987; Alexander et aL, 1988; Damsma et al., 1988b; Carter et al., 1988; Reid et al., 1988; Robinson and Whishaw, 1988; Rollema et aL, 1988; Hurd et al., 1988; Ton et aL, 1988; Wood et aL, 1988; Zetterstr6m et at., 1988; Bean et al., 1989; Hurd and Ungerstedt, 1989a; Hurd and Ungerstedt, 1989b; Hurd and Ungerstedt, 1989e; Hurd and Ungerstedt, 1989d; O'Connor et al., 1989; Westerink and de Vries, 1989; Westerink et al., 1989a; Westerink et al., 1989b.

Noradrenaline L'Heureux et al., 1986; Routledge and Marsden, 1987; Abercrombie et al., 1988; Egawa et al., 1988; Kal6n et al., 1988b; Vathy and Etgen, 1988.

AcetylchoHne Ajima and Kate, 1987; Console et al., 1987; Damsma et al., 1987a; Damsma et at., 1987b; Damsma et at., 1988a; Damsma et al., 1988c; Herreras et al., 1988; Westerink et al., 1989b.

Serotonin Kate et al., 1986; Kal6n et al., 1988a; Sharp et al., 1989.

Adenosine Ballarin et al., 1987; Brodie et al., 1987.

Amino acids Bite et al., 1986; Delgado et al., 1972; Lehmann and Hamberger, 1983; Sandberg and Lindstr6m, t983; Jacobsen and Hamberger, 1984; Lehmann and Hamberger, 1984; Lerma et al., 1984; Lehmann et al., 1985b; Lerma et al., 1985; Sandberg et al., 1985; Tossman and Ungerstedt, 1985; Jacobsen et al., 1986; Solis et al., 1986; Tossman et al., 1986a; Tossman et al., 1986b; Young and Bradford, 1986; Bradford et al., 1987; Lehmann, 1987; Mufioz et al., 1987; Brodin et al., 1988; Wade et al., 1988; Men6ndez et al., 1989.

Peptides Brodin et al., 1983; Lindefors et al., 1985; Lindefors et al., 1986; Brodin et al., 1987; Maidment et al., 1989.

Metabolism Zetterstr6m et al., 1982; Hutson et al., 1985; Kuhr et al., 1988; Kuhr and Korf, 1988; Schasfoort et al., 1988; Korf, 1989.

Microdialysis methodology Johnson and Justice, 1983; Justice et al., 1983; Delgado et al., 1984; Ungerstedt, 1984; Jacobsen et al., 1985; Korf and Venema, 1985; Hernandez et al., 1986; Lerma et al., 1986; Tossman and Ungerstedt, 1986; Wages et al., 1986; Westerink and Tuinte, 1986; Benveniste et aL, 1987; Church and Justice, 1987; L6nnroth et al., 1987; Sandberg et al., 1987; Sharp et al., 1987a; Ungerstedt and Hallstr6m, 1987; Alexander et al., 1988a; Benveniste and Diemer, 1988b; Kendrick, 1988; Sells et al., 1988; Westerink and De Vries, 1988; Amberg and Lindefors, 1989; Benveniste, 1989; Benveniste et al., 1989a; Donzanti and Yamamoto, 1989; Lehmann, 1989; Lindefors et al., 1989; Moghaddam and Bunney, 1989; Reiriz et al., 1989; Lehmann and Sandberg, 1990. r

MICRODIALYSIS----THEORY AND APPLICATION 199

TABLE 2.

Gastrointestinal tract Heart Eye Adrenal gland Blood

Subeutis

Skeletal muscle

Meirieu et al., 1986 Hamberger, 1989 Ben-Nun et al., 1988 Medvedev et al., 1989 Collin and Ungerstedt, 1988; Hurd et al., 1988; Speciale et al., 1988; Arner et aL, 1989 Bito et al., 1966; Delgado et al., 1972; L6nnroth et aL, 1987; Arner et aL, 1988 Lehmann, 1989

Vezzani et al. , 1988; Benveniste et al., 1989; Hurd and Ungerstedt, 1989)].

Interstitial fluids are generally characterized by a very low protein content (see review by Aukland and Nicolaysen, 1981) and therefore never added to perfusion fluids. Furthermore, adding proteins to peffusion fluids would defeat one of the main advantages of microdialysis which is the ability to analyze dialysis substance concentrations directly by high performance liquid chromatography (HPLC) without prior deproteinization. However, in some studies 0.2-0.8% bovine albumin is added to the perfusion medium (Brodin et al., 1983, 1987; Kendrick et aL, 1986, 1988) to avoid peptide 'stick- ing' to the membrane, plastic- and stainless steel

tubing and thus suboptimal recovery values (see also Section 5.4).

In summary, it seems prudent--for microdialysis experiments in brain t issue--to use fluids that contain an identical ionic composition as that of the brain interstitial space and further if one of these ions is under investigation and a substitution is required then to ascertain whether or not this affects control conditions,

4. MEASUREMENTS WITH MICRODIALYSIS

Bito et aL (1966) were the first who tried to measure substance concentrations in the brain interstitial

TABLE 3. PERFUSION FLUID COMPOSITIONS (IN mM UNLESS OTHERWISE INDICATED)

Distilled water

Saline: 0.9% NaC1 Saline: 0.9% NaC1; 1.85% CaC12 (pH 7.2) Saline: 0.9% NaC1; 0.5% bovine serum albumin

Ringers solution

134 NaC1; 5.9 KC1; 1.3 CaC15 1.2 MgC12 (O2-sat.) 147 NaCl; 2.4 CaC12; 4 KC1 (pH 6.0) 147 NaCl; 3.4 CaCl2; 4 KC1 (pH 6.1) 145 NaC1; 1.3 CaCl2; 4 KC1 (pH 7.2) 155 NaCl; 5.5 KCI; 2.3 CaC12 189 NaC1; 3.9 KCl; 3.4 CaC12 (pH 7.2)

Modified Ringers solution

145 NaC1; 2.7 KCl; l MgCI2; 1.2 CaCl:; 0.2 ascorbate (pH 7.4)

Buffered Ringers solution

147.2 d NaCl; 3.4 CaCl,; 2.76 KC1; 1.16 MgCl2; 0.62 K2HPO4; 113.5/aM ascorbic acid (pH 6.9)

Krelm Ringer solution

147.2 NaC1; 4.0 KCI; 3.4 CaCI 2 (pH 6.1) 138 NaCl; II NaHCO3; 5 KCI; I NaH2PO4; 1 CaClz; l MgC12; II glucose (pH7.5)

Krebs Ringer bicarbonate

122 NaCI; 3 KC1; 1.2 MgSO4; 0.4 KHzPO4; 25 NaHCO3; 1.2 CaCI 2 (pH 7.4)

Krebs--Henseleit bicarbonate buffer

118.4 NaC1; 4.7 KC1; 0.6 MgSO4; 2.5 CaC12; 1.2 NaH2PO4; 25 NaHCO3; 11 glucose

Mock-¢erebrospinui fluids

120 NaCI; 15 NaHCOa; 5 KC1; 1.5 CaCl:; 1.0 MgSO4; 6 glucose (pH 7.4) 126.5 NaC1; 27.5 NaHCO3; 2.4 KCI; 0.5 KH2PO4; 1.1 CaCl2; 0.85 MgC12; 0.5 Na2SO4; 5.9 glucose (pH 7.5) 127 NaC1; 2,5 KCI; 1.3 CaC12; 0.9 MgCl: (pH 6.0) 127 NaC1; 2.5 KC1; 1.3 CaCI2; 2 MgC12 132.8 NaCl; 3.0 KCI; 24.6 NaHCO3; 6.7 urea; 3.7 glucose 135 NaC1; 3.0 KC1; 1.2 CaCl2; 1.0 MgCl2; 2.0 phosphate (pH 7.4) 140 NaCI; 3.0 KC1; 2.5 CaCl2; l MgCl:; 1.2 Na2HPO4; 0.27 NaH2PO4; 7.2 glucose (pH 7.4) 150 NaCI; 3 KC1; 1.7 CaCI2; 0.9 MgC12 119.5 NaC1; 4.75 KC1; 1.27 CaCl:; 1.19 KH2PO4; 1.19 MgSO4; 1.6 Na, HPO 4 (pH 7.2)

JPN 35/3---B

200 H. BENVENISTE and P. C. Hf2TTEr, mlER

],4-

1.2-

1.0-

0.8£

0.4~

0.2 t 0

mM Ca+* in Interstltlalspace of rat cortex

,o'oo ' 20'°0

distance (l~m) between electrode tip and dialysis membrane

FIG. 4. The impact of dialysis with calcium-free fluid on the interstitial milieu in the vicinity of the probe. Perfusion of the probe (2.5/~l/min) was started 1 hr following implantation into the cortex. The interstitial Ca 2+ concentration was measured 30 min after the perfusion start at various dis- tances from the perfused dialysis probe by a Ca2+-sensitive microelectrode. Each point on the curve represents one experiment. Data from Benveniste et al., 1989a. For further

details see Sections 3 and 5.

space using dialysis. They collected samples from dialysis membrane bags filled with 6% dextran in saline chronically implanted in dogs (the system was not perfused in any way). Ten weeks later the bag was removed and the fluid analyzed for its contents of amino acids and ions. The K ÷ concentration was found to be 4.71raM. Measurements with ion- selective microelectrodes have found brain interstitial K* concentration to be only 3.2raM, sO the bag f luid--or dialysates--apparently did not represent correct interstitial concentrations. In 1972 Delgado and co-workers presented data from a continuously perfused microdialysis probe. Results were given only as dialysate substance concentrations and no attempts were made to transform these into 'true' brain interstitial substance concentrations. Obvi- ously, because the microdialysis probe produces a dialysate of fluid from the interstitial space the substance concentrations measured herein can only be reflections of true brain interstitial substance concentrations. ZetterstrSm et al. (1982) were probably the first who attempted to calculate brain interstitial concentrations from dialysate concentrations using a simple in vitro calibration of the microdialysis probe in water.

4.1. CONCENTRATION MEASUREMENTS BASED

ON IN VITRO CALIBRATION

According to Zetterstr6m et al. (1982) the ratio between the concentration of a substance in the outflow solution and the undisturbed* concentration of the same substance in the solution outside the

* It is important to note that recovery measurements are not evaluated from the substance concentration in the immediate vicinity of the dialysis machine which/s disturbed due to extensive drainage (cf. Section 5.1).

probe is defined as 'recovery' and expressed either as a ratio or as a percentage. Recovery would normally be determined with the probe in an aqueous solution containing a known concentration of the substance in question. The probe is continuously perfused at a constant flow rate with a fluid devoid of the substance and samples are collected during fixed times intervals. The recovery of the particular substance is then calculated as:

Recovery,, ,,,o = Cout/Ci

where (?out is the concentration in the outflow solution and Ci the concentration in the medium.

Having determined recovery for the substance in question, dialysate substance concentrations are transformed into brain interstitial concentrations using the following formulae:

= ~ou~/Recoveryi, vitro" (1)

Ci is the substance concentration in the brain interstitial space and Cou , is the concentration of the dialysate (Fig. 5). Unfortunately, this simple calculation is not valid because mass transport of substances in brain tissue is different from that of aqueous solutions (see Section 5.5). This means that calculated interstitial concentrations based solely on dialysate concentrations and in vitro recovery--for most substances--underestimate true interstitial concentrations. In the following section we will discuss additional factors that must be taken into account when dialysate concentrations are transformed into brain interstitial concentrations.

5. FACTORS AFFECTING SUBSTANCE COLLECTION WITH MICRODIALYSIS

5.1. PERFUSION FLOW RATE AND TIME

Figure 6 (upper part) illustrates that recovery for a given rnicrodialysis probe increases when perfusion flow rates are kept low and furthermore that recovery changes with time; initially, recovery is high, then rapidly decreases and levels off although it never quite reaches steady state. The slight decrease of recovery after 30--60 min of perfusion is neglected for practical purposes and this part of the curve is used for determinations as described in Section 4.1. It has generally been assumed that the high dialysate substance concentrations immediately after implantation represented a traumatic tissue response [i.e. a local break-down of blood-brain barrier (BBB) and cell membranes]. Although this may play an important role (cf. Section 6) it should be kept in mind that the same time-dependent decrease of recovery is found in aqueous solutions (Delgado et al., 1984; Amberg and Lindefors, 1989; Benveniste et al., 1989; Benveniste, 1989). This is probably due to a steep concentration gradient across the dialysis membrane when the probe is first inserted into the medium which then gradually declines due to intense substance drainage in the immediate vicinity of the membrane (of. Fig. 4). The theory behind both the establishment and time- dependency of concentration profiles in the immediate vicinity of a dialysis membrane has recently been explored by Amberg and Lindefors (1989).


I, Com

Cout Re¢°verYln vitro" C

out

out

Rec°verYin vitro

FIG. 5. Estimation of brain interstitial concentrations from in vitro calibration.

5.2. FLux

In contrast to recovery, the flux of substances (the amount harvested by the membrane per unit time) across the dialysis membrane increases with higher flow rates. However, as illustrated in Fig. 6 (lower part), flux is not significantly influenced by perfusion rates above 5-10/~l/min (there is some variability with different probe designs). This is most likely caused by diffusion limitation (the concentration gradient across the dialysis membrane is near maxi- mum at these particular flow rates). Alternatively, a stagnant flux with higher flow rates could also be due to a positive hydrostatic pressure gradient across the membrane which may occur with a rigid tube design and lead to a decrease of mass transport into the probe (Johnson and Justice, 1983). High flow rates with microdialysis are best avoided because the perfusion fluid leaves the probe under pressure and may cause tissue damage---this is exactly the major draw- back with the push-pull perfusion technique (see reviews by Ugerstedt (1984) and Benveniste (1989)). The actual flow rates at which significant filtration forces build up across the dialysis membrane are unknown at present and likely influenced by the probe design. Recently, net fluid exchange during microdialysis (in both serial and parallel probe designs) was measured during perfusion flow rates of 2/~l/min and 10/~l/min, respectively. In both instances fluid loss before and after perfusion was less than 0.1% (Lindefors et aL, 1989) suggesting that these flow rates can be used safely.

5.3. TF_~PERArUR~S AND CnANO~ IN OUTER SUBSTANCE CONCENTRATION

Wages et al. (1986) found that in vitro recovery of 3,4-dihydrophenylacetic acid (DOPAC) at a flow rate of 0.2 #l/min increased approx. 30% when the tem- perature was increased from 23°C-37°C. These re- suits have since been confirmed by others (Benveniste et al., 1989; Lindefors et al., 1989) and are not unexpected because diffusion coefficients are known to increase 1-2%°C (Bard and Faulkner, 1980). Therefore,/n vitro probe calibration should always be performed at temperatures identical to the tissue (Zetterstr6m et aL, 1982; Korf and Venema, 1985; Van Wylen et al., 1986; Wages et al., 1986; Young and Bradford, 1986; Benveniste et al., 1989; Benveniste, 1989).

Recovery is independent of changes in the outer (undisturbed) substance concentration (Ungerstedt et al., 1982; Hamberger et aL, 1983; Johnson and Justice, 1983; Sandberg and Lindstr6m, 1983; Ungerstedt, 1984; Tossman and Ungerstedt, 1986; Kendrick, 1988). This is fortunate, because if the opposite were true as pointed out by Johnson and Justice (1983) microdialysis would obviously not be able to measure interstitial concentration changes correctly. When the outer substance concentration is suddenly increased or decreased, the concentration gradient across the dialysis membrane changes corre- pondingly thus keeping recovery constant. The fact that recovery is independent on changes in the outer substance concentration should not be confused with

202 H. BENVENIST~and P. C. HOTTEMEm_R

A I=

t o

is

10

0i . 4 8 12 16

recovery %

E 411

2O

0 0

Recovery %

10

8

6 ~

4

2

0 5 10 15 20

Flux (dpm/rnin)

FtG. 6. Upper part: Recovery of 14C-mannitol. A typical curve of recovery as a function of perfusion flow rate (11) and time (@), respectively. Recovery is inversely related to perfusion flow rate and time, respectively. When perfused at a constant flow rate, relative recovery is initially high but decreases rapidly. The recovery at individual perfusion flow rates was measured after 90 min of perfusion. Data from Benveniste et al., 1989a. Lower part: In contrast to recovery values, the net flux across the dialysis membrane is only slightly influenced by perfusion rates above 5pl/min- 10#l/min (varies with microdialysis probe designs). Data from Benveniste eI al., 198%. For further details see

Section 5.

recovery and time-dependency (cf. Section 5.1). The latter phenomenon is also dependent on concentration gradient changes but here the 'undisturbed' outer concentration does not change correspondingly and recovery therefore decreases.

5.4. DIALYSIS MEMBRANE AREA AND COMPOSITION

Recovery is directly proportional to the size of the dialysis membrane area (Hamberger et al . , 1983; Sandberg and Lindstr6m, 1983; Ungerstedt, 1984; Tossman and Ungerstedt, 1986). This is advan- tageous when one wishes to detect brain interstitial substances that are present in very low concentrations. By increasing the membrane area low concentrations can be detected with reasonably high perfusion flow rates while maintaining an adequate time resolution.

Dialysis membrane materials are known to interact with transported substances via unknown mechanisms and affect mass transport. For instance, recovery of acidic amino acids is lower than that of the neutral ones (Sandberg and Lindstr6m, 1983). Another extensive study examined how different dialysis membrane materials affected in vi tro recovery measure-

ments (Kendrick, 1988). It was found that recovery for certain substances varied as much as 20% between different probes (Kendrick, 1988). Recently, the 'adhesiveness' of the dialysis membrane was quantified by a 'capture probability constant', which according to the authors should be used cautiously, because the 'capture' may be a time-dependent, saturable phenomenon (Lindefors et al., 1989).

5.5 . DIFFUSION COEFFICIENT

It is known that the diffusion coefficient is inversely proportional to the size of the substance in question, i.e. solute radius (for futher details see Friedman, 1986). Consequently, diffusion coefficients will be lower for substances with higher molecular weights which may help explain the inverse relationship between recovery and molecular weight (Kendrick, 1988; Benveniste et al., 1989). Because this is found even with dialysis membranes characterized by high cut-offs (50,000) (Kendrick, 1988) diffusion (for molecular weights up to 5000 daltons) is not likely to be limited by membrane pore size.

Recovery~,~t,o is known only for a small number of substances and is--with few exceptions--less than that found in simple, aqueous media* (L6nnroth et al., 1987; Arner et al., 1988; Benveniste et aL, 1989; Lindefors et al., 1989). This is because diffusion coefficients in complex media (i.e. the brain is a complex medium comprising the interstitial-, the intracellular- and the vascular compartment) are lower when compared to those in aqueous solutions. Diffusion of most hydrophilic substances is impeded by impermeable cell membranes (Nicholson et al., t979; Nicholson and Rice, 1986) and mass transport is confined to the interstitial space (ct) which comprises only 20% of the total brain volume (Levin et al., 1970; Nicholson et al., 1979; Nichoison and Phillips, 1981). Several studies illustrate the import- ance of taking these factors into account. Lazarewicz et al. (1986) measured the Ca 2÷ concentration in dialysates collected from a horizontal tube implanted in rabbit hippocampus during perfusion with a Ca 2+- free medium. With 20% in vi tro recovery, the brain extracellular Ca 2÷ level was calculated to be approx. 0.75 mM. This is considerably below the known extracellular Ca 2+ concentration in brain, i.e. 1.2-1.3 mM (see also Vezzani et al., 1988). In another study in vi tro recovery of adenosine changed from 20.3% at a flow rate of 2/~l/min to nearly 100% when the flow rate was decreased to 0.1/~l/min (see also Section 5.1). When repeated in rive, the respective adenosine concentrations recovered in dialysates increased from 0.12-1.26 #M. The calculated 'interstitial' adenosine concentration based on in vitro recovery, amounted to 0.12/0.20, or 0.60 #M, but should in fact have been twice as high (i.e. approx. 1.26 with 100% recovery)

* For substances like K ÷ and glycerol the in vivo recovery equals that of in vitro (Arner et al., 1988; Benveniste et al., 1989; Lindefors et al., 1989). This is in accordance with previous investigations showing that K + in brain rapidly traverses the cell membrane due to rapid exchange mechanisms (Nicholson et al., 1979; Nicholson and Phillips, 1981). Furthermore, glycerol--a lipophilic molecule---is not very likely to be impeded by the presence of cell membranes.

MICRODIALYSIS---THEoRY AND APPLICATION 203

(Van Wylen et al., 1986). Recently, Alexander et al. (1988) demonstrated the significant difference between in vitro and /n vivo recovery measurements using tritiated water (THO). For a given microdialysis probe and perfusion flow rate (above 0.2/zl/min) in vitro THO recovery was always higher than that in vivo.

6. EFFECT OF MICRODIALYSIS ON THE BRAIN MICROENVIRONMENT

6.1. BRAIN COMPARTMENTATION

The brain comprises three different water spaces: the intracellular, the interstitial and the vascular space (Fig. 3). The determination of substances in the brain interstitial space with microdialysis obviously requires that the implantation procedure leaves the boundaries between these spaces intact. Blood-brain barrier integrity has been measured after implantation of a microdialysis probe with BBB-impermeable substances such as ~-amino-isobutyrate and sodium technitate (Benveniste et al., 1984; Tossman and Ungerstedt, 1986). Despite some degree of brain tissue trauma with the insertion procedure (see below) it was nevertheless found that the BBB around the microdialysis probe was intact shortly (30 min-2 hr) after implantation. Previously, Hamberger and co-workers (1983) and Hamberger and Nystr6m (1984) compared amino acid concentrations in blood, CSF, and brain tissue (homog- enates) with concentrations in dialysates 24 hr after probe implantation. 'Interstitial' amino acid concentrations (calculated according to Eqn. 1) were dis- tinctly lower than those of blood and tissue but correlated remarkably well with CSF values. These results suggested that the BBB was intact. There are as yet no reports on BBB integrity in chronic preparations > 1 day).

6.2. TISSUE REACTIONS

It is important to know the time-course of tissue changes after implantation of the probe, because the trauma itself, the inflammatory changes and subse- quent gliosis and fibrosis may inflict severe changes of local brain metabolism and blood flow. In addition, tissue diffusion characteristics may also change. Tissue reactions were studied after implantation of a serial microdialysis probe in rat hippocampus (Benveniste and Diemer, 1988). Within two days the tissue adjacent to the membrane (a 50#m border zone) exhibited edema, minor hemorrhages and accumulations of polymorphonuclear leukocytes. Eosinophilic neurons, indicating cell death, were occasionally present 100-150#m from the implant. Otherwise, normal neuropil surrounded the microdialysis membrane. After three days there was astrocyte hypertrophy followed by connective tissue displacement after 2 weeks. The latter was still present after 60 days (see also Hamberger and NystrSm, 1984). These results were largely confirmed by Shuaib et aL (1990) using the silver degeneration staining method. They further demonstrated that degenerating axons were present both adjacent to the implantation site and also in remote brain areas

such as the corpus callosum and contralateral hippocampus.

Tissue reactions are classic and unavoidable in the vicinity of both implants and lesions (Del Rio Hotega and Penfield, 1927; Stensaas and Stensaas, 1976; Collias and Manuelidis, 1957). In view of this, the reliability of chronic microdialysis measurements becomes questionable (to be discussed further in Section 6.5).

6.3. LOCAL CEREBRAL BLOOD FLOW AND 2-DEOXYGLUCOSE PHOSPHORYLATION

4-Iodo[N -methyl- ~4C]-antipyrine autoradiographs --illustrating regional cerebral blood flow--from rat brains with a borizontally placed microdialysis probe in hippocampus demonstrate that immediately (within I hr) after implantation hippocampal blood flow is reduced by 40% (Benveniste et al., 1987). At the same time local cerebral blood flow (LCBF) is decreased by 60% in remote brain regions such as striatum, lateral septum and thalamus. 2-Deoxy-D- [14C]glucose (2DG) autoradiographs reveal two kinds of changes within 3 hr after implantation: (a) Distinct regions with increased 2-DG uptake in close prox- imity to the dialysis membrane, and (b) a general reduction in 2-DG uptake in both hippocampus and remote brain regions. LCBF and local cerebral 2DG phosphorylation (LCMR~) were near normal in all brain regions 24 hr after implantation.

All these experiments have since been repeated using a vertical probe design (Sandberg and Benveniste, 1989). Hippocampal CBF was unchanged 2 hr after insertion whereas LCMR~ was moderately increased (approx. 50% of the values obtained with the horizontal probe). Importantly, there were no remote changes of either blood flow or glucose consumption.

It is well-known that brain lesions cause a metabolic deactivation of those brain areas anatomically connected with the lesioned region (Feeney and Baron, 1984). This phenomenon is termed 'diaschisis'. However, the changes of LCMR~ and LCBF found in remote brain areas after implantation of the dialysis membrane were widespread and transient and therefore uncharacteristic of diaschisis (Feeney and Baron, 1984). Alternatively, these findings could be explained by the induction of a spreading depression (SD) (Leao, 1944) which is known to cause widespread transient changes of LCBF and LCMRac (Shinohara et al., 1979; Lauritzen, 1987). In experiments where a microdialysis probe was combined with a calcium-sensitive microelectrode there were transient changes of the DC-potential and the interstitial Ca 2÷ concentration characteristic of a SD (Fig. 7) (Benveniste et al., 1989). In cortex, a SD is characterized by depression of evoked and spon- taneous EEG activity and a negative shift of the DC-potential (Nicholson and Kraig, 1981; Hansen, 1985). This in turn is associated with changes in the distribution of ions between the intra- and extracellular compartments: Interstitial potassium concentration increases and those of calcium, sodium and chloride decrease (Hansen, 1985). Most ion concentrations spontaneously revert to normal after 30 sec-1 min.

204 H. BENVENISTE and P. C. H(YrrEMEER

mM Ca +'~

12

05 ~

O1

3 min

FIG. 7. Elicitation of a spreading depression. The arrow indicates the time of insertion of the microdialysis probe. Data from Benveniste et al., 1989a. For further information

see Section 6.

6.4. USE OF TETRODOTOXIN AND CALCIUM DEPLETION FOR ESTIMATING FUNCTIONAL

DAMAGE AFTER IMPLANTATION Westerink and De Vries (1988) evaluated func-

tional tissue damage after probe implantation using tetrodotoxin (TTX) and calcium-free perfusion fluids. In normal brain tissue both maneuvers would abolish transmitter release and for instance dopamine should not appear in the dialysates*. Acutely and several hours after implantation of a parallel designed probe (o.d. 800/~m), dopamine release was not inhibited by calcium depletion and only moderately so by TTX. When a transtriatal dialysis probe (with a smaller outer diameter) was used acutely, dopamine output displayed some calcium and TTX dependency. When tested after 24 hr dopamine output was completely abolished with both probe designs suggesting a return to normal tissue function--this is in accord with the normalization of cerebral blood flow and 2-DG phosphorylation that also occurs at this time.

6.5. THE POSSIBLE INFLUENCE OF THE DISTURBED BRAIN MICROENV1RONMENT ON RESULTS

OBTAINED BY MICRODIALYSIS

Based on histological, functional, metabolic and blood flow changes caused by the probe insertion it appears that the optimal time for commencing microdialysis is 8-48 hr after implantation. At this time local blood flow and glucose metabolism is minimally

* It is well-known that calcium ions play a fundamental role in the process of 'excitation-secretion coupling' in neurochemical transmission. Release of transmitter occurs as a result of the arrival of an action potential at the nerve terminal. The depolarization phase of the action potential, results in an opening of voltage-dependent Ca 2 + channels in the presynaptic membrane. The influx of Ca 2+ down its electrochemical gradient through calcium channels leads to a transient rise in intracellular Ca 2+ which triggers a transient release of the transmitter. The Ca 2+ entry into the presynaptic terminal can be divided into two phases, i.e. the early phase which is abolished by tetrodotoxin (TTX) (which blocks sodium-dependent voltage-operated channels) and the late phase which is unaffected by TTX but can be inhibited by Mn 2+ and Co 2+. Consequently, TTX, inorganic Ca 2+ blockers or calcium depletion inhibit transmitter release (cf. review by T6r6k, 1989).

disturbed, the tissue releases transmitter(s) (dopamine) in a voltage-dependent manner and chronic tissue reactions have not yet developed. However, this optimal time is often either impractical or un- obtainable. This is particularly so in experiments where microdialysis measurements are combined with electrophysiological recordings. Futhermore, there is still some controversy as to whether experiments carried out with acute or chronical implanted probes give similar substance concentration measurements. Some data but not all show that control levels of amino acids and biogenic amines and changes of these induced by various stimuli differ between acute and chronic preparations. Korf and Venema (1985) found a steady increase in the resting release of several amino acids over a period of 9 days. The same authors also found that responses to concentrated potassium infusions and electroconvulsive shock differed with time. A similar trend has been observed by Westerink and Tuinte (1986) who found that potassium-induced dopamine release disappeared 3 days after implantation whereas amphetamine- induced dopamine release was still present but dimin- ished 8 days after implantation (see also Roltema et aL, 1988). In contrast, Delgado et al. (1984) found near-constant concentrations of aspartate, threonine and glutamate over a period of 10 months. Addition- ally, Strecker et al. (1986) using dialysis membrane of smaller dimensions than those of Westerink and De Vries (1986), demonstrated an almost complete inhibition of dopamine release after apomorphine in an acute implantation situation.

7. SOLUTIONS TO THE PROBLEM OF DETERMINING BRAIN INTERSTITIAL

CONCENTRATIONS WITH MICRODIALYSIS

We previously discussed that the calculation of 'true' brain interstitial substance concentrations based exclusively on dialysate concentrations and in vitro recovery leads to significant underestimations due to the fact that mass transport of substances in vivo differs from that in vitro (of. Sections 4.1 and 5.5). Other approaches are therefore required and those that have been used so far can be divided into two categories: (a) Methods that operate without time resolution, i.e. perfusion flows are either very low (or non-existent), or the calculation requires repetitive measurements which means that substance concentration fluctuations during some short experimental intervention cannot be detected, and (b) methods that allow a continuous transformation of dialysate concentrations without a loss of time resolution.

7.1. METHODS WITHOUT TIME RESOLUTION According to Fig. 6, major reductions of perfusion

flow rates result in recovery values that approach 100%. This would in theory eventually produce dialysate concentrations equal to those of the brain interstitial space. Actually Bito et al. (1966) were the first who accomplished this with an unperfused implanted dialysis bag. The dialysate concentrations they obtained are probably slightly overestimated-- for instance resting glutamate concentrations


amounted to 450/~i (Sections 4, 8.1 and 8.2). Wages et al. (1986) measured dopamine in dialysates at flow rates of 0.1 and 2/~l/min, respectively, and found that dialysate dopamine concentrations decreased from 9 to 2 ma. Similar results were found for adenosine by Van Wylen et al. (1986).

Jacobson et al. (1985) solved diffusion equations and found that recovery depended on perfusion flow rate, membrane area, and an 'average mass transfer coefficient'. From their equation and with the use of regression analysis they could estimate extracellular concentrations. Interstitial concentrations derived from the curve fit were only slightly higher than those based on in vitro recovery measurements. The equation assumed a constant concentration profile outside the probe and furthermore a dialysis membrane that constituted a diffusional resistance. How- ever, both assumptions are probably wrong. First, because the microdialysis probe constantly drains substances from the brain, tissue diffusion gradients change correspondingly (become less steep with time) and lead to a reduction of mass transport (cf. Section 5.1). Secondly, dialysis affects substance concentrations quite far from the probe as shown in Fig. 4, and not only in its near vicinity. This is evidence that the membrane does not constitute a major diffusion barrier and may therefore be regarded as part of the medium.

Lerma et al. (1986) measured amino acid concentrations in dialysates collected from a dialysis membrane loop in which the perfusate circulated for several hours. Assuming that compounds crossed the dialysis membrane by simple diffusion, amino acid concentrations were calculated by computerized non- linear regression analysis of the dialysis data at different circulation times. For instance resting glutamate concentrations were found to be 2.9/~i which is surprisingly low considering the fact that others have measured much higher levels (Young and Bradford, 1986; Globus et al., 1988; Faden et al., 1989) even though dialysates were not recircled. Unfortunately, Lerma et al. (1986) did not test the validity of their method by measurements of ion concentrations in the dialysate as other investigators have done (Bito et al., 1966; Benveniste et al., 1988, 1989) and the results are therefore difficult to evaluate.

L6nnroth et al. (1987) presented yet another method that in principle would produce a 'true' interstitial concentration of any compound in the brain----or in any tissue for that matter. The dialysis membrane--implanted in subcutis--was perfused with perfusion media containing glucose in different concentrations. Using regression analysis they determined the exact point at which no net flux of glucose occurred across the membrane. This glucose concentration would be in equilibrium with that of the surrounding tissue and hence equal to it.

Lindefors et al. (1989) recently presented a somewhat similar approach as Lfnnroth and co-workers (1987) by infusing the substance of interest via the microdialysis probe and calculating recovery as follows:

c~° - C~n_~ Recovery = (2)

c~°

where Cm is the substance concentration in the perfusion medium, and Ci,-o,t is the concentration after passage through the medium. Having determined recovery /n vivo the interstitial concentration (Ci) could be calculated from Eqn. 1 (Section 4.1). The problem with this approach is, as pointed out by the authors, that recovery determinations using Eqn. 2 cannot discriminate between what is actually transported through the membrane and what may be adhering to it. This appears for some unknown reason to be a more significant problem for recovery measurements determined by Eqn. 2 than with Eqn. 1. Some substances 'stick' to the membrane (cf. Sections 3.3 and 5.4) and there is, for example, a discrepancy between in vitro recovery of sucrose determined from Eqns 1 and 2, respectively (Lindefors et al., 1989).

7.2. METHODS WITH TIME RESOLUTION

Benveniste et al. (1989) calculated interstitial substance concentrations assuming that mass transport of substances between the probe and the tissue only occurs by diffusion*. In a simple medium (i.e. without cells), the flux J measured in relation to the total area is given by Ficks law as:

t3C J = - O - - (3)

dr

where D is the diffusion coefficient, C is the substance concentration in a unit volume and r is the distance measured perpendicular to the area considered. In the case of a tissue with a total volume of V and interstitial space volume V0, the flux J to the probe is:

l = - ~D --OC (4) t~r

where ct = V o / V , 15 is the diffusion coefficient in the tissue interstitial space. It is assumed that cells do not exchange the substances of interest with the interstitial space. These theoretical considerations show that mass transport or flux in vivo is always smaller than in vitro because ~t is less than 1 and/~ less than D. The so-called tortuosity factor (As= D/I~) de- scribes the increased diffusion pathway in vivo due to the presence of impermeable cell membranes (Safford and Bassingthwaighte, 1977). Assuming that diffusion around the probe is axisymmetrical and strictly radial (for a more extensive mathematical analysis see Benveniste et al., 1989) Eqn. 4 can be

* It is well-established that mass transport in brain interstitial space occurs both by diffusion and convection, i.e. convection fmass transport caused by fluid motion (Bradbury et al., 1981; Cserr et al., 1981; Szentistv~.nyi et al., 1984). However, we will not consider the latter process because diffusion is usually the dominant mass transport mechanism in the interstitial fluid. Nevertheless, when cali- brating the microdialysis probe/n vitro, we have to avoid convection which can easily be introduced by stirring. Convection is avoided if the aqueous solution is solidified with agar (0.2-1.0%).

solved and the undisturbed (cf. Section 4.1) brain interstitial substance is found:

8"

Ci = [K*A21ot] x ~'o~t/Recovery~n ,,,,o. (5)

Equation 5 states that the interstitial concentration can be calculated by inserting in vitro recovery, ~'out and K22/cc The inclusion of the interstitial volume fraction a and the tortuosity factor 2 in the equation is not surprising and was pointed out by Nicholson and Phillips (1981) who described diffusion from a point source in brain tissue. A comparable situation prevails when substances move in the interstitial space towards the microdialysis probe. Diffusion is retarded by the tortuous interstitial space and the amount of substance reaching the probe is reduced by the volume fraction. They determined the value of 22/ct for tetramethylammonium (TMA, an extracellular marker in brain tissue) using iontophoresis and ion-selective microelectrodes (Nicholson et al., 1979; Nicholson and Phillips, 1981) and found an extracellular volume fraction of 0.2 and a tortuosity factor of 1.6 which would give 22/ct value of appoximately 12. A similar value was found for Ca 2+ using microdialysis (Benveniste et al., 1989). The ratio of 12 for Ca 2÷ is consistent with the microelectrode study of Nicholson and Rice (1986), who showed that Ca 2÷ diffusion in brain interstitial space is similar to that of TMA, and with that of Safford and Bassingthwaighte (1977), who showed that 4SCa2+ mainly diffuses in the interstitial space of myocardial tissue.

Therefore for these calculations one needs to know both the outflow concentration and the in vitro recovery of the substance at 37°C and also the diffusion characteristics in vivo (i.e. ct, 2 and K). It is worth emphasizing that the use of Eqn. 5 is only valid if the surrounding microenvironment is inactive. The effects of cellular uptake, exchange mechanisms and metabolism of a substance on ~t, ). and K are not fully understood at present and only a few transmitters and ions have been characterized in this respect (Rice et aL, 1985; Nicholson and Rice, 1986). Considering all these theoretical aspects and the many uncertain- ties involved it is obvious that the determination of

*When determining brain interstitial concentrations according to Eqn. 5 with microdialysis it is also necessary to incorporate an additional factor, K. The factor K, given by:

i L expresses the differences between substance concentrations on the inside of the dialysis membrane, when dialysis is performed in simple, Cb, and complex, ~'b media, respectively. If mass transport in brain tissue equaled that of an aqueous solution, then Cb = Cb, or K = 1. However, measurements of TMA concentration profiles in red blood cell suspensions (RBCs) and aqueous solutions, respectively, have shown that Cb > ~'b (Fig. 8), which indicates that diffusion in water is driven by a smaller concentration gradient across the dialysis membrane compared with diffusion complex media. We may interpretate this as if recovery in water is likely to be underestimated because diffusion in this particular medium is sufficiently fast to counteract the sink action of the microdialysis probe (Benveniste et al., 1989).

t Diffusion coefficient of calcium.

0 0

4

3= 3

i .

lime/min

206 H, BENVENISTE and P. C. HfdTTEMEIER

FIG. 8. The time-course of glutamate concentration changes in dialysates collected in CAI of hippoeampus before, during and after 10 min of ischemia. The dark and white arrow indicate the onset of ischemia and recirculation, respectively. Data from Benveniste et aL, 1989b. For further

information see Section 8.

interstitial concentration with microdialysis using Eqn. 5 can only be approximated.

Lindefors and co-workers (1989) presented a solution where the interstitial concentration (~'i) was found to be related to the dialysate concentration (Cout) in the following way:

Cou~q (6) Ci = 4zcLa/3 h(t /3/R 2)

where q is the perfusion flow rate, L the dialysis membrane length, ,t the interstitial volume fraction, /3 the diffusion coefficient in brain tissue. The term h(t) is a function of the scaled, non-dimensional time, t = t t / 3 / R 2, R is the radius of the dialysis probe [for details of the mathematical analysis see (Arner et al., 1989)].

The diffusion coefficient in brain tissue is, as previously discussed, determined by:

/3 = D i ). 2.

Inserting this expression into Eqn. 6 we get:

_ qCout 22 Ci = 4x-~-~ ~ t ) ' (7)

As in Eqn. 5, the interstitial substance concentration Ci is directly proportional to 22 and inversely proportional to cc Therefore, knowledge of the in vivo diffusion characteristics is also required for this equation. This is partly circumvented by calculating the diffusion coefficient in vivo using the following formula:

/3 = Recovery/, vivo x D (8) Recoveryi, vitro X O~

where Recoveryi, vwo is determined from Eqn. 2. To test the validity of Eqn. 6 we can calculate

the interstitial Ca 2+ concentration using data from Benveniste et al. (1989): q = 5/~i/min = 8.3 x 10-" m3/sec; Cout=0.0078 mM; L = 2 x 10 -3m; /3 = D/22 = 0.79 x 10-9t/1.62 = 3.08 x 10 -1° m2/sec; ct = 0.2 (Nicholson et al., 1979); h(t) = h ( t t D / R 2) = h(5400 sec x 3.08 x 10 -l° mZ/sec/(260 x 10 -6 m) 2) = h(24.6) = 0.23 (Amberg and Lindefors, 1989; Lindefors et al., 1989), the diffusion coefficient of calcium in vitro is 0.79 x 10-gm2/sec (Hille, 1984)

M1CRODIALYSIS~THEORY AND APPLICATION 207

and inserting these parameters into Eqn. 6 the interstitial Ca 2+ concentration amounts to:

8.3 x 10 -H m3/sec x 0.0078 mM ~ i = 4 n x 2 x 10 -3 m x 0 .2 = 1.81 raM.

x 3.08 x 10-1°m2/sec x 0.23

which is somewhat higher than that found by ion-selective microelectrodes (1.2-1.3 mM) (Hansen, 1985). It appears that the use of Eqn. 6 tends to overestimate brain interstitial substance concentrations (see Sections 8.1 and 8.3).

In the next section we will transform glutamate and dopamine dialysate concentrations into interstitial concentrations using both Eqns 5 and 6 for comparison.

daltons). Rice et al. (1985) determined the diffusion coefficient for dopamine in brain tissue to be 0.68 x 10-t°m2/sec.

According to Eqn. 6 the interstitial glutamate concentration is:

0.441~M X 8.3 x 10 -H m3/sec ~'~i 4 x it x 2 x 10 -3 m x 0.2 x 0.68 345/~M.

X 10-1°m2/sec x 0.31

where Co,t = 0.44/~M, q = 5 #1/rain = 8.3 x 10 -11 m3/ see, L = 2 x 10 -3 m (Co,t, q and L originate from data by Benveniste et al., 1989), D = 0.68 x 10 -~° m2/ sec (Rice et al., 1985), h ( t ) = 0.31 (Lindefors et al., 1989) and g = 0.2 (Nicholson et al., 1979).

8. TRANSFORMATION OF GLUTAMATE AND DOPAMINE DIALYSATE

CONCENTRATIONS INTO BRAIN INTERSTITIAL CONCENTRATIONS

8. I. NORMAL BRAIN TISSUE

8.1.1. Glutamate

Equation 5 transforms dialysate concentrations into brain interstitial concentrations but does n o t - - as previously stressed---consider transmitter uptake and/or metabolism. Bradford et al. (1987) demonstrated that glutamate concentrations in dialysates increased by 75% when a glutamate uptake inhibitor was added to the perfusion medium. Therefore, unless this uptake is corrected fo r prior to using Eqn. 5, interstitial (synaptic) glutamate concentrations would probably be underestimated:

Corrected dialysate glutamate concentrat ion= dialysate glutamate concentration × correction factor

~out = 0.44 ]IM X 1.75 = 0.77 #M.

[0.44gM is the average dialysate concentration of glutamate in rat CAI hippocampus at a perfusion flow rate of 5 #l/rain (Benveniste et al., 1989).] Insert- ing this value in Eqn. 5:

Ci = [(K x 22)Ict)] x (Coot/Recoveryi, vi~,o),

we arrive at an interstitial concentration of:

Ci = [(0.7 x 1.62)/0.2] x (0.77 #M/0.05) = 138 #M

where K =0.7 (Benveniste et al., 1989), 2 = 1.6 (Nicholson and Philips, 1981), ct =0 .2 (Nicholson and Philips, 1981) and the recovery~, v~,ro of glutamate at 37°C is 5% with a perfusion flow rate of 5/d/rain (Tossman and Ungerstedt, 1986; Benveniste, 1989). It is important to note, that our use of 2 and ~t found for TMA, assumes that diffusion characteristics for glutamate and TMA, respectively, are similar.

Unlike Eqn. 5, Eqn. 6 by Lindefors et al. (1989) apparently does not require a correction for transmitter uptake (see Lindefors et aL, 1989). Unfortunately, the diffusion coefficient for glutamate in brain tissue is unknown and no attempts have been made to calculate this value according to Eqn. 8. In the following, therefore, we will assume that the diffusion coefficient for glutamate is similar to that of dopamine which is known (the molecular weights of dopamine and glutamate are quite similar, 156 vs 169

8.1.2. Dopamine

In order to calculate interstitial dopamine concentrations with Eqn. 5 we will use data from Hurd and Ungerstedt (1989d). They measured basal striatal concentrations of dopamine in rats before and after systemic administration of cocaine which inhibits dopamine high affinity uptake (see review by Hurd, 1989). Dialysate concentrations of dopamine increased from 10.6 to 45 nM during cocaine administration. Thus, having corrected for uptake we can now use Eqn. 5:

Ci = [(K x 22)/~] x (Co,t/Recovery~, vii,o)

and we get

45nMX 1.62x0.7 Ci = = 2.2 ]2M

0.2 x 0.24

where Co,t = 45 nm, ,2. = 1.6, K =0.7, ~t = 0.2 and 0.24 represents the recovery~ vi,ro for dopamine, when a 4 mm CMA/10 dialysis membrane is perfused at 2 #1/min (Collin and Ungerstedt, 1989; Hurd and Ungerstedt, 1989d). Once again it is important to note that our use of 2 and ~t found for TMA, assumes that diffusion characteristics for dopamine and TMA, respectively, are similar.

Interstitial dopamine concentrations calculated from Eqn. 6 yields

10.6riM x 3.3 x 10 - l l •i = 4 x T r x 4 x l 0 _ 3 m x 0 . 2 =3 .3gM

X 0.68 x 10-1°m:/sec x 0.31

where ~'o,t is 10.6nM, q = 3.3 x 10 -llm3/sec, L = 4 x 10 -3 m (~'o,t, q and L originate from data by Hurd and Ungerstedt, 1989),/) = 0.68 x 10 -l° m2/sec (Rice et al., 1985), h(t) = 0.31 (Lindefors et al., 1989) and ~t = 0.2.

8.2. INTERPRETATION OF CALCULATED INTERSTITIAL GLUTAMATE AND DOPAMINE CONCENTRATIONS

OBTAINED BY MICRODIALYSIS

Results from the previous section suggest that calculations of glutamate and dopamine interstitial concentrations from Eqns 5 and 6 correlate reasonably well. As anticipated both methods provide concentrations that are several-fold higher than values obtained by using the simple/n vitro recovery formula (Eqn. 1). How do we assess the accuracy of these determinations? This is not possible for glutamate

208 H. BENVENISTE and P. C. HOTTEMEmR

because there are no standards to compare with. For dopamine there appears to be a good correlation between microdialysis calculations and measurements performed with carbon fiber microelectrodes (Moghaddam et al. , 1987). On the other hand is it at all relevant from a biological standpoint to be concerned with the accuracy of these microdialysis determinations? Neurotransmitter concentrations are obviously not constant but fluctuate within a few hundred microseconds as a result of release and uptake (Alberts e t al. , 1983). Therefore microdialysis with its limited time resolution cannot measure actual transmitter concentration changes in the synaptic cleft during neurotransmission but only an average of these. This is important to keep in mind when evaluating experimental data. A good example is the apparent paradox that the rather high resting glutamate concentrations we find in v ivo are potentially neurotaxic in v i t ro (Choi et al. , 1987; Frandsen and Schousboe, 1987; Frandsen et al. , 1989; Rosenberg and Aizenman, 1989). However, with reference to our previous arguments we may assume that neurons are not constantly exposed to these 'high' average glutamate concentrations and therefore not damaged.

In the following section we will determine interstitial (synaptic) glutamate concentrations during transient global ischemia. In contrast to normal tissue this situation is characterized by electrical silence, no transmitter uptake and therefore a more stabile transmitter concentration which can be calculated and evaluated by Eqns 5 and 6, respectively.

8.3. GLUTAMATE CONCENTRATIONS IN ISCHEMIC BRAIN TISSUE

Glutamate may play an important role in the pathogenesis of selective neuronal injury and death following transient cerebral ischemia (Jargensen and Diemer, 1982; Wieloch, 1985a; Rothman and Olney, 1986; Greenamyre, 1986; Auer and Siesj6, 1988; Choi, 1989). In short the excitotoxin hypothesis explains selective neuronal death induced by transient global ischemia as a result of an abnormally high concentration of glutamate present in the synaptic cleft during ischemia. This triggers excessive and uncontrolled Ca 2÷ influx in the postsynaptic neurons that ultimately causes cell death. Microdialysis has been crucial for the verification of this hypothesis. Figure 8 demonstrates the time-course of glutamate concentration changes in dialysates collected from the rat CA1 hippocampal region before, during 10 min of ischemia and upon recirculation (Benveniste e t al., 1989b). Glutamate concentrations increase in the dialysates during ischemia in all animals. These results have largely been confirmed by several other investigators. Hagberg et al. (1985) demonstrated a 3.5-fold increase of glutamate during 10min of ischemia in the rabbit hippocampus, Globus et al. (1988a) and Korf e t al. (1988) found a 7-fold release of glutamate during 20 min of ischemia in rat striatum, Shimada et al. (1989) demonstrated a 5- to 10-fold increase of glutamate in cat cortex and finally Hillered e t al. (1989) measured (>40.fold increase) of glutamate in striatum after permanent occlusion of the middle cerebral artery.

The calculation of 'true' glutamate concentrations during ischemia requires as previously discussed that glutamate dialysate levels are transformed into brain interstitial concentrations. This is first done using Eqn. 5. High affinity glutamate uptake is non- functional during ischemia (Barbour et aL, 1988) and therefore any corrections for this effect is unneces- sary. The mean glutamate concentration measured in dialysates during 5-10rain of ischemia is 2.7/~M (Benveniste et al. , 1989). The Recoveryinoi,, o of glutamate is 5% (Tossman and Ungerstedt, 1986) and the interstitial space size is known to shrink during ischemia by 50% to 0.1 (Hansen and Olsen, 1980). The tortuosity factor has not been determined during ischemia and we must therefore use the value of 1.6 obtained in normal brain tissue (Nicholson et al., 1979; Nicholson and Rice, 1986).

Inserting all these parameters into Eqn. 5 we arrive at an interstitial glutamate concentration of:

Ci = 0.7 x 0.62/0.1) × 2.7#u/0.05 = 968/~M

which is still somewhat underestimated because the tortuosity factor may increase during ischemia (Nicholson and Rice, 1986).

Glutamate concentrations evaluated by Eqn. 6 yield:

•i = 2.7/ZM x 8.3 x 10-11 m3/sec =4.2ram 4 x n x 2 x 10-3m× 100.1 x 0.68 x 10-1°m:/sec x 0.31

where ~'out = 2.7/~M, q = 8.3 x 10 -H m3/sec, L = 2 x 10 -3 m (Benveniste e t al. , 1989), ~ = 0.1 (Hansen and Olsen, 1980), D =0.68 × 10-1°m2/sec (Rice et al. , 1985) and h(t)=0.31 (Lindefors et al., 1989).

It is noteworthy that the simple recovery formula (Eqn. 1), would only have given a glutamate concentration of 2.7/,u/0.05 = 54 # u which appears to represent a 20- to 80-fold underestimation of the 'true' interstitial (synaptic) glutamate concentration calculated from Eqns 5 and 6. This is important for the verification of the excitotoxin hypothesis because glutamate concentrations of 1--4 mM are definitely toxic during a brief exposure whereas concentrations in the low micromolar range require much longer time to produce neuronal injury (Choi et al. , 1987; Frandsen and Schousboe, 1987; Frandsen et al. , 1989; Rosenberg and Aizenman, 1989).

9. CONCLUSION

The microdialysis technique is an important break- through in the neurobioiogical sciences and has pro- vided much needed information on the complex functions of the intact brain. The real benefit of expanding the applications to include other organs and tissues remains to be seen but preliminary results seem promising. However, despite its increasing pop- ularity, microdialysis is still far from being the routine method it was perhaps intended to be. In the past, much work has necessarily gone into improving the technology involved whereas less attention was focused on the many methodological problems and limitations that could lead to erroneous data interpretations and conflicting results. Hopefully, the preced- ing chapters have clearly illustrated that this trend is


changing and that an increasing effort is directed towards finding solutions to the many data analytical problems that are still partially unresolved.

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microdialysis—theory and application

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