Microdialysis in the Neurosciences: Techniques in the Behavioral and Neural Sciences

Netherlands

Introduction to microdialysis

Outline

Introduction to intracerebral microdialysis

Microdialysis compared with other in vivo release models

Chapter 1

Introduction to intracerebral microdialysis

Urban Ungerstedt*

*Correspondence to: Dr. U. Ungerstedt, Department of Pharmacology, Karolinska Institute, Stockholm, Sweden

Publisher Summary

This chapter discusses the concept of intracerebral microdialysis. The idea of microdialysis is to mimic the passive function of a capillary blood vessel by perfusing a thin dialysis tube implanted into the tissue. Microdialysis is a technique for both recovering and administering substances in a tissue. The major features of microdialysis include: (1) it samples the extracellular fluid; (2) it can be performed locally in almost every organ and tissue of the body; (3) it can be performed in the intact tissue of the living, awake, and freely moving animal, distinguishing it from other preparations; and (4) it can be used for recovering and/or introducing endogenous and exogenous substances in the tissue. In essence, a microdialysis probe is a push-pull cannula with a dialysis membrane applied over its tip. Microdialysis has a real potential for studies in peripheral organs, including blood. Microdialysis is also finding its use in nonmammalians, such as the Lamprey and in plants. Microdialysis can easily bridge the gap between the animal model and man because of its simplicity and limited invasiveness. Microdialysis is becoming a promising tool in pharmacokinetic and drug distribution studies in animals as well as man.

1 Development of microdialysis

Analyzing the biochemical functions of the body has commonly involved the sampling of blood or dissection of tissue. However, the blood is a distant reflection of the many events taking place in cells and organs and the dissected tissue presents a static picture, mixing the chemistry of organelles, cells and extracellular fluid. The chemical interplay between cells occurs in the extracellular fluid but this compartment is usually overlooked due to its experimental inaccessibility.

There have been many experimental attempts to approach the extracellular environment of the intact brain, for example ventricular pefusions, push pull cannulae (Gaddum, 1961) and cortical cup perfusions. However, it seems that the idea of introducing a dialysis membrane into the tissue has provided the first generally applicable way of interacting with the extracellular compartment.

Bito et al. (1966) were the first to implant dialysis sacs containing 6% dextran in saline into the subcutaneous tissue of the neck and into the parenchyma of the cerebral hemispheres of dogs. Ten weeks later they removed the sacs surgically and analyzed their content of amino acids. These experiments introduced the idea of a compartment surrounded by a dialysis membrane which equilibrates to the extracellular environment. However, the intention was not to follow changes over time but to avoid physiological fluctuations and reach an average concentration of the substances of interest.

Delgado et al. (1972) developed a dialytrode which was very similar to the present microdialysis probes consisting of two stainless steel tubes soldered together forming a push-pull cannula ending in a small permeable bag. The dialytrode was tested in vivo in monkeys. Delgado’s original vision is approaching reality today: This system may provide a new diagnostic and therapeutic procedure in man, to obtain neurochemical information from, and to deliver drugs to specific structures of the brain.

In our own laboratory we got the idea of microdialysis while mapping the monoamine neurons under the fluorescence microscope. We saw dopamine diffusing from nerve terminals in the immediate vicinity of blood vessels. It occurred to us that thin dialysis tubes, so-called hollow fibres, would function like blood vessels and be devoid of a blood brain barrier if they were implanted into the brain. We implanted them by drilling holes in each temporal bone of rats and guided the fibre through the brain. When dopamine neurons were labeled by perfusing with [³H]dopamine we could monitor baseline and amphetamine stimulated release in anaesthetized animals. In freely moving animals the release of [³H]dopamine was correlated with behavioural activation (Ungerstedt and Pycock, 1974). Nieoullon et al. (1977) were perfecting the use of [³H]tyrosine labeling with push pull cannulae at this time and it seemed of little interest to repeat their studies using microdialysis. We therefore looked for an analytical method sensitive enough to detect endogenous dopamine and found it in the developing LC EC (Kissinger et al. 1973; Mefford, 1981). We designed the loop probe and a concentric probe (e.g., see Fig. 1 in Chapter 4) to avoid the trauma caused by the horizontal implantation and combined the use of these probes with LC EC detection of endogenous dopamine (Ungerstedt et al., 1982; Zetterström et al., 1983; Ungerstedt, 1984; Tossman and Ungerstedt 1986).

2 Features of microdialysis

The idea of microdialysis is to mimic the passive function of a capillary blood vessel by perfusing a thin dialysis tube implanted into the tissue. The concentration of compounds in the perfusate, determined with appropriate analytical techniques, thus reflects the composition of the extracellular fluid due to the diffusion of substances back and forth over the membrane. In sum, microdialysis is a technique for both recovering and administering substances in a tissue. The major features of microdialysis are:

— It samples the extracellular fluid, as distinct from the whole tissue collected by biopsies, punches or dissections (See Chapter 2).

— It can be performed locally in almost every organ and tissue of the body (including blood).

— It can be performed in the intact tissue of the living, awake and freely moving animal, distinguishing it from other preparations such as slices and synaptosomes.

— It makes it possible to sample continuously for hours or days in a single animal, which in addition to other advantages, decreases the number of animals needed in an experiment.

— It can be used for recovering and/or introducing endogenous and exogenous substances in the tissue. It is, for example, possible to give a drug systemically while estimating the local drug concentration, the local biochemical effect in addition to the resulting physiological and behavioural response (see Chapters 6 and 7).

— Microdialysis collects a representative sample of all substances in the extracellular fluid (provided that they pass the membrane), carries them out of the body and makes them accessible to conventional analytical techniques. This distinguishes the technique from in vivo sensor techniques such as implantable biosensors and in vivo electrochemistry (see Chapters 2 and 5).

— The ultimate sensitivity and time resolution of the substances recovered is determined by the analytical technique (Chapters 2, 5 and 6).

— Experimental studies indicate that microdialysis causes minimal damage to the blood-brain barrier (Benveniste et al. 1984, Tossman and Ungerstedt 1986). This makes it possible to: (a) study drug penetration into the brain by analyzing the drug concentration in the perfusate; and (b) compare the effects of drugs on the brain when applied directly via the microdialysis probe or via the systemic route.

3 Comparisons to push-pull perfusions

Most of the above features of microdialysis are shared with the push-pull perfusion technique (Chapter 2). In essence, a microdialysis probe is a push-pull cannula with a dialysis membrane applied over its tip. This may seem as a small difference, however, it adds several interesting features to the technique:

— It simplifies perfusions. Since the flow is unidirectional there is no need to balance the flow. In a push pull cannula too much push infuses liquid into the brain and too much pull sucks brain tissue into the cannula. The simplicity is of particular advantage when using awake, freely moving animals when long tubings and movements of the animal make it difficult to balance the flow.

— It minimizes damage to the tissue, because there is no direct contact between the liquid flowing inside the membrane and the cells of the tissue.

— The membrane excludes large molecules from diffusing into the perfusate. This protects certain molecules from being broken down by tissue enzymes once they are recovered from the tissue. In addition, the process of microdialysis purifies the sample making it possible to introduce it directly into the analytical instrument, for example an HPLC system, even when used on line with the experiment (Chapters 5 and 6).

— It introduces a sterility barrier between the perfusion liquid and the brain tissue. The microdialysis probe only needs to be sterile on the outside, which is easy to achieve by placing it in an alcohol solution. This is an advantage during chronic experiments in large animals, susceptible to infections, and diminishes the need to use sterile perfusion liquid.

— The size of the perfused area can be influenced by varying the length of the membrane.

— Microdialysis can be performed in blood or in body cavities such as the uterus, the peritoneal cavity and the mouth which is impossible with an open push-pull system.

— The performance of a microdialysis probe and its membrane can be studied in vitro. Different probes can be compared as regards the recovery of a particular substance or a single probe can be studied for its ability to recover substances with different molecular weight or charge, (e.g., Chapters 12–14).

4 Principles of microdialysis

Microdialysis is in principle a very simple technique. A tubular dialysis membrane is introduced into the tissue or placed into contact with a moist surface, for example, a mucous membrane. The tube is perfused with a liquid which equilibrates with the fluid outside the tube by diffusion in both directions. The degree of equilibration is subject to known laws of physical chemistry.

The complexity of the technique comes from interactions between the dialysis tube, the perfusion liquid and the living tissue. In order to understand how to perform microdialysis, and how to interpret the results, it is important to picture the many events taking place during this interaction.

4.1 Tissue disturbance, damage and gliosis

There is a rapid fall in the concentration of most substances in the perfusate during the first 30 minutes of microdialysis, which is probably due to both an initial lesion of the tissue causing an excessive release from cellular storage compartments, and the establishment of a new steady state level of most extracellular substances because of the drainage through the probe (Lazarewicz et al., 1986; Benveniste et al., 1989; Amberg and Lindefors, 1989; also see Chapter 9). In our experience the degree of initial damage is influenced by the operating procedure and care should be taken to introduce the probe slowly into the brain.

There is also an initial period of disturbed tissue function involving increased glucose metabolism, decreased blood flow, disturbed transmitter release etc. after implanting a microdialysis probe. This period lasts from around 30 min to 24 h depending upon which function one measures and which substance one analyzes (Benveniste et al., 1987; Drew et al., 1989; Osborne et al., 1990, Osborne et al., 1991). In analogy with slice experiments physiological and pharmacological experiments are usually started when the substance of interest has reached a baseline level in the perfusate (cf., Chapter 9). Several control experiments have been developed to determine the validity of the data generated from various types of microdialysis studies (see below).

We assume that the initially high levels are due to damage and that the decrease in levels of substances in the perfusate is due to a normalization. This interpretation seems valid as transmitters, which are stored in high intracellular concentrations, are particularly high initially while metabolic substances are less so. Another observation supporting the theory is that baseline is reached quicker in larger brains where the relative size of the probe is smaller. In the human brain this often occurs within 10–20 min (Meyerson et al., 1990). Changes in substances that are not stored in high intracellular concentrations, for example metabolites, probably reflects a complex series of events leading to the establishment of a new equilibrium (see Chapter 9).

The amount of trauma associated with the introduction of the microdialysis probe probably determines the time it takes for substances to reach baseline and how soon they react as expected to various manipulations such as omitting calcium or blocking sodium channels. This may be related to differences in neuronal sensitivity for alterations in, for example, energy production

A related problem is for how long a time substances can be reliably recovered once they have reached a baseline. This varies between substances as well as investigators (Westerink and Tuinte, 1986; Westerink and De Vries, 1988; Osborne et al., 1990 Chapter 9). The differences are not surprising in view of biological differences in synthesis, storage, metabolism, density of innervation, delicacy of nerve terminals, regional difference in blood flow and presence of potentially neurotoxic substances. There are also obvious differences between laboratories and their type of probe, use of guide cannula, maintenance of sterility, implantation procedures, composition of the perfusion medium, biocompatibility of membrane, species etc etc.

In view of this complexity it is probably not wise to discard the difficulty in maintaining constant recovery conditions over long periods as solely a problem of gliosis formation (Chapter 9 for further discussion). In fact, few studies have been done on the formation of glioses with proper histochemical techniques (Hamberger and Nyström 1984, Benveniste and Diemer, 1988; Juhasz et al., 1989). In all probability the potential of microdialysis to recover substances over long periods of time is still an open question.

4.2 Diffusion and recovery

The microdialysis probe communicates with the extracellular fluid by the principle of diffusion along a logarithmic concentration gradient towards and away from the probe. The direction of this gradient depends upon the composition of the perfusion fluid.

The composition of the perfusion liquid dominates the immediate environment of the probe. If, for example, calcium is excluded from the perfusion liquid the extracellular fluid surrounding the probe is depleted of calcium, impairing synaptic transmission (Imperato and Di Chiara, 1984; Westerink et al., 1988). Conversely, including a substance in the perfusate will make it spread from the probe along a concentration gradient which is opposite to that of the calcium depletion. This means that the composition of the perfusate should be as close as possible to normal physiological levels of the most essential compounds in the extracellular environment (Moghaddam and Bunney, 1989). How far this similarity should be extended has to be determined by the individual experiment as the very purpose of a microdialysis experiment is to remove or administer substances by using a dissimilarity between the perfusion liquid and the extracellular fluid.

The recovery of substances from the extracellular fluid depends on the length of the dialysis membrane, the flow of the perfusion liquid, the speed of diffusion of the substance through the extracellular fluid and to a lesser extent the properties of the membrane (Amberg and Lindefors, 1989; Benveniste, 1989; Lindefors et al., 1989; Bungay et al., 1990). The speed of diffusion is a property related to the diffusion through the parenchyma as well as to the active elimination from the tissue by uptake into cells and blood capillaries (Chapters 3, 4 and 7). Recovery is the same in both directions across the membrane (Amberg and Lindefors, 1989).

For small molecules, such as the monoamine transmitters, the factor limiting recovery is usually the speed of diffusion through the extracellular fluid, not the diffusion through the membrane (Amberg and Lindefors, 1989), although for neuropeptides the membrane can also be an important variable (Chapters 12–14). In the tortuous brain parenchyma the speed of diffusion may be around 50% of that in water, while the corresponding figure for the membrane may be as high as 80%. Although it is tempting to think that the membrane represents an important barrier it is actually the ability of a substance to diffuse through the extracellular space that determines both the amount of substance that can be recovered and the speed by which a change can be detected (Chapters 3 and 4).

It is convenient to distinguish between relative recovery (concentration recovery) and absolute recovery (mass recovery) (Ungerstedt 1984, Chapter 7). Relative recovery is the concentration of a particular substance in the perfusate when it leaves the probe expressed as a per cent value of the concentration in the surrounding medium. Absolute recovery is the total amount of the substance recovered during a defined period of time, expressed in moles/liter. When a microdialysis probe is tested in vitro, relative recovery decreases and absolute recovery increases as the perfusion flow is increased. Both values reach a plateau when the diffusion speed through the surrounding medium reaches its maximum.

In vivo the relative recovery is constant as long as the perfusion conditions remain the same. However, the absolute recovery of a substance varies with its production/release in the tissue. The main reason for performing microdialysis is usually to follow this change in the extracellular levels of endogenous or exogenous substances.

The fact that the speed of diffusion in the extracellular fluid determines recovery means that two substances, with different diffusion characteristics, may appear in the perfusate in a concentration ratio that is different from that in the tissue. This may be the case with dopamine and DOPAC, where dopamine probably is recruited from the immediate vicinity of the probe because of the efficient reuptake mechanism that exists for dopamine. In contrast, DOPAC is diffusing into the perfusate from a larger area than dopamine causing a larger DOPAC/dopamine ratio than expected (Sharp et al., 1986).

4.3 Neurotransmitter release

A central question is whether recovered transmitters are reflecting true synaptic release or a more unspecific overflow from synaptic and non synaptic sources. Blocking the dopamine (Westerink et al., 1987), 5-HT (Kalén et al., 1988a) or noradrenaline (Kalén et al., 1988b) reuptake mechanisms increases the level of the transmitters in the perfusate, and stimulating dopamine autoreceptors (Zetterström and Ungerstedt, 1984) gives the expected decrease of dopamine. Perfusion with TXX, in order to block sodium channels, lowers acetylcholine (Damsma et al., 1987), dopamine (Westerink and De Vries, 1988) and noradrenaline (Kalén et al., 1988b). The omission of calcium lowers dopamine (Imperato and Di Chiara, 1984), 5-HT (Kalén et al., 1988a) and acetylcholine (Marien and Richard., 1990) levels. Although these data strongly suggest that the microdialysis probe recovers an overflow from synaptic release, it does not prove that this overflow is quantitatively related to synaptic release.

Studies of GAB A are more complicated. Uptake block increases GAB A in the perfusate (Kehr and Ungerstedt 1988), whereas the effect of low calcium and TTX varies dependent upon the implantation procedure, how soon after the implantation of the probe the experiment is performed and if the animal is anaesthetized or conscious. Drew et al. (1989) and Westerink and De Vries (1989) did not find any effects of TTX in acutely implanted animals while Osborne et al. (1990) were able to demonstrate decreased GABA after both TTX and omission of calcium in awake animals implanted with the microdialysis probes for 1–4 days.

The correct interpretation of the GABA experiments is probably that GABA in the perfusate does not originate solely from synaptic sources. However, the fact that GABA in the perfusate is increased by dopamine D1 receptor agonists and lowered by D2 agonists (Reid et al., 1990) is positive evidence for release under synaptic control.

An important matter to consider when perfusing with, for example, TTX and high potassium is that these substances influence all neurons in the area. A change in the release of one transmitter may therefore be the result of an effect of TTX itself, as well as secondary effects mediated by changes in the release of other transmitters. It seems even conceivable that different effects can cancel each other out.

4.4 The true extracellular concentration

Another fundamental problem is the relationship between the concentration in the perfusate and the concentration in the extracellular fluid. Several attempts have been made to determine this concentration by extrapolating from in vitro recovery values, but there is now general agreement that this is not correct because of the difference in diffusion coefficients between water and tissue (Chapters 3 and 4).

The best estimate of extracellular concentrations is obtained from in vivo experimerits (Chapters 3, 4 and 7). It can be calculated by varying the perfusion flow during an in vivo experiment, measuring the change in the substance of interest coming out of the probe and then extrapolating to zero flow (Jacobson et al., 1985) or by perfusing with varying concentrations of the substance and then calculating the equilibrium concentration, i.e. the concentration at which the substance in the perfusate does not change during the perfusion because it has the same concentration inside the probe as in the extracellular fluid (Lönnroth et al., 1987). Both these in vivo methods require that the concentration in the extracellular fluid remains constant during the length of the experiment.

A third, rather elegant method, is to use a reference substance in the perfusate (Chapter 7) or perfuse with it before the systemic injection of an exogenuous compound. The method is based on the fact that the recovery over the membrane is the same in both directions. Given that diffusion properties of the reference substance in the perfusate and the substance to be recovered from the brain are the same, the per cent loss of the reference substance from the perfusate will be the same as the per cent recovery of the substance from the brain. This recovery value is then used to calculate the concentration in the extracellular fluid. Thus, in pharmacokinetic experiments the true extracellular concentration of a drug, can be determined by equilibrium dialysis during steady state or by including a reference substance in the perusate. This substance should have the same diffusion characteristics as the systemically administered drug, ideally appear as a peak in the same chromatographic system and be pharmacologically inactive in the concentration used.

The fact that the concentration in the perfusate is related to the diffusion coefficient in the extracellular fluid means that a change in this coefficient during the experiment may be wrongly interpreted as a change in extracellular concentration. This may happen if edema develops in the tissue, for example, during experimental ischemia or spreading depression. However, it can be controlled by including a reference substance in the perfusate. This may be a labeled tracer dose of the same substance one is recovering from the brain. A change in this substance will reveal a change in the diffusion coefficient and can be used as a correction