Extracellular Space

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The extracellular space comprises about 18 to 20% of the total tissue volume of most soft organs. This tissue compartment represents an important link between blood capillaries and cell soma for transport of molecules in both directions. The space is filled with the extracellular fluid (ECF) which, in turn, directly communicates with other body fluids such as lymph or cerebrospinal fluid (CSF). The ECF is a glutinous buffered medium consisting of salts and low molecular weight compounds, as well as polysaccharides, glucoproteins, polypeptides, and enzymes forming a complicated extracellular matrix. In the brain, released neurotransmitters can diffuse from the synaptic cleft into the larger extracellular compartment or can be involved in slower signalling over longer distances, so called volume transmission (Bjelke et al., 1994). Because bioanalytical techniques such as homogenization mix cytosol with ECF, it is impossible to use these methods to analyze the ECF contents. In contrast, the microdialysis probe enables "eavesdropping" on intercellular chemical communications.

6.1.3 Microdialysis Probe

The concept of in vivo sampling by microdialysis is based on the construction of a special cannula—a microdialysis probe—which, once implanted in the tissue, will more or less mimic the function of a blood vessel. Just as vessels serve for blood circulation, the probe is perfused with a physiological solution. In the same way that many molecules can pass through the walls of blood capillaries, the probe is provided with a semipermeable membrane allowing free diffusion of low molecular weight compounds. Microdialysis probes are constructed in several different types, among them a concentric design of a perfusion cannula, which is probably the most commonly used in experimental research. The typical examples of such a probe type depicted in Figure 6.1 represent some of the commercially available microdialysis probes. The CMA Model 10 features a "classical" construction of a probe for small and medium-sized laboratory animals, whereas a later modification (CMA 11) allows for microdialysis in very small areas of the brain. For applications in peripheral tissues, where the use of a flexible cannula is desirable, the CMA 20 is recommended.

All three probes shown in Figure 6.1 are similar in design: the inlet and outlet lines consist of two concentric cannulae, where the thinner line is longer and extends into the semipermeable membrane at the tip of the probe. The membrane is glued at its proximal end into the outer shaft of the probe; at its distal end, it is plugged with a glue. Alternatively, it can be glued to the inner cannula (CMA 10), as well, to strengthen the whole construction. Typically, the inner tube serves as the inlet and the outer tube as the outlet for the perfusion medium, but reversal of the flow is also possible. The membrane lengths for most of the commerical probes vary from 1 mm to several centimeters, and the outside diameters range from 0.2 to 0.7 mm.

Several membrane materials are suitable for the construction of microdialysis probes: cuprophane (regenerated cellulose), polycarbonate, polysulfone, polyacrylonitrile. Molecular cutoffs of these membranes are usually in the range of 5000 to 20,000 Da for sampling of small molecules, whereas for large and lipophilic compounds, cutoffs from 20,000 to 100,000 Da are available. Ideally, the membrane should function only by dialysis, not ultrafiltration. This means passage of components only by equilibrating concentration gradients, not by allowing passage of fluid through the membrane due from a high pressure gradient or through large pores in the membrane. Such ultrafiltration phenomena may cause serious disturbances of the tissue homeostasis and reduce dialysis recovery.

inlet outlet steel shaft membrane

Figure 6.1 Microdialysis probes based on the concentric assembly of the inlet and outlet tubes provide the highest mechanical strength, as well as homogeneous diffusion paths from the surrounding environment; they also cause the lowest tissue damage. The probes differ mainly in the construction of the inlet/outlet lines, as seen when comparing these constructions. The shaft and the capillaries can be either rigid and made of metal (A) or fused silica (B) or flexible and made of polyurethane (C). The outer diameter of the membrane on the CMA/11 probe (B) is 0.24 mm, whereas the membranes of CMA/10 and CMA/20 probes (A and C) are 0.5 mm in diameter.

6.1.4 Dialysis Recovery

Since the first reports on microdialysis in living animals, there have been efforts to estimate "true" (absolute) extracellular concentrations of recovered substances (Zetterström et al., 1983; Tossman et al., 1986). Microdialysis sampling, however, is a dynamic process, and because of a relatively high liquid flow and small membrane area, it does not lead to the complete equilibration of concentrations in the two compartments. Rather, under steady state conditions, only a fraction of any total concentration is recovered. This recovery is referred to as relative or concentration recovery, as opposed to the diffusion flux expressed as absolute or mass recovery. The dependence of recovery on the perfusion flow rate is illustrated in Figure 6.2. As seen, relative recovery will exponentially decrease with increasing flow as the samples become more

16 |xl/min

Figure 6.2 In vitro recovery versus flow rate for a typical microdialysis probe (CMA/ 10, 4 mm polycarbonate membrane, cut off 20,000 Da) in a quiescent medium and at ambient temperature. Typical flow rates used from brain microdialysis applications are in the range of 0.1 to 5 /iL/min.

pmol/10mln iry 16

16 |xl/min

Figure 6.2 In vitro recovery versus flow rate for a typical microdialysis probe (CMA/ 10, 4 mm polycarbonate membrane, cut off 20,000 Da) in a quiescent medium and at ambient temperature. Typical flow rates used from brain microdialysis applications are in the range of 0.1 to 5 /iL/min.

and more diluted, while the mass recovered per time unit will increase and reach a plateau.

Relative recovery can be mathematically expressed by Fick's law of diffusion as modified by Jacobson (Jacobson et al., 1985). In this relationship, recovery is the ratio of the concentration in the perfusate to the concentration extracellular. For the mass recovery, an expression similar to a MichaelisMenten formula for enzymatic reactions was derived (Ekblom et al., 1992). A number of other mathematical models for quantitative microdialysis have been proposed and are reviewed elsewhere (Justice 1993; Kehr, 1993b).

6.2 TECHNICAL ASPECTS OF MICRODIALYSIS 6.2.1 Microdialysis Instrumentation

The microdialysis probe is the heart of the method, as a chromatographic column is the heart of the HPLC instrument. Rigid CMA probes, Models 10, 11, and 12, are used for stereotaxic implantations into the brain, where the probe can be fixed (cemented) to the skull. A flexible probe design (CMA 20) allows the placement of such a catheter into the moving tissues (muscle) or peripheral organs for studies in freely moving animals. The technical difficulties of microdialysis experiments impose requirements for precise liquid delivery, minimized dead volumes, and the capability of handling small sample volumes.

A representative arrangement for microdialysis sampling in anesthetized rats is shown in Figure 6.3. The microinjection pump should include features such as a pulse-free electronically controlled dc motor and should be precali-

Microinjection Pump Cma 200

Figure 6.3 Basic setup for microdialysis experiments on small laboratory animals. The system can he used for simultaneous collection from up to three microdialysis probes. The main components are (A) CMA/100 Microinjection Pump, (B) CMA/140 Microfraction Collector, (C) temperature controller, (£>) liquid switch for switching between different perfusion fluids, and {£) in vitro stand for storage of microdialysis probes.

Figure 6.3 Basic setup for microdialysis experiments on small laboratory animals. The system can he used for simultaneous collection from up to three microdialysis probes. The main components are (A) CMA/100 Microinjection Pump, (B) CMA/140 Microfraction Collector, (C) temperature controller, (£>) liquid switch for switching between different perfusion fluids, and {£) in vitro stand for storage of microdialysis probes.

brated for precision glass syringes to ensure smooth fluid flows in the range of from 1 nL/min to 1 mL/min. In the arrangement shown, three syringes can be used simultaneously, either for three separate microdialysis probes or for one probe (when switching between different perfusion media is required for stimulations, drug delivery, etc.). Switching between lines must not be allowed to introduce air bubbles into the system.

When not in use, microdialysis probes should be stored wet and protected from mechanical damage. Also, testing the probes by in vitro recovery is easily accomplished if the probes are fixed in an appropriate stand. Fractions can be collected manually, but it is more convenient to use fraction collectors capable of collecting fractions as low as 1 ¡xL.

Several commercial constructions are available for triple (CMA 140, Fig. 6.3B) or dual (CMA 142} probe collecting and with refrigeration (CMA 170) for collecting easily degradable compounds. An alternative is direct coupling of the microdialysis probe's outlet to the high pressure injection value (CMA 160) of an HPLC system (Johnson and Justice, 1983). This enables on-line analysis of samples, with the only delay due to the analysis time of a particular separation method. A rapidly growing interest in clinical applications of microdialysis led to the construction of a special portable microdialysis pump and a flexible microdialysis catheter for use in adipose tissue and noncon-tracting muscle in humans (Fig. 6.4), Samples are collected manually by using a specially designed microvial.

6.2.2 HPLC Analysis

Essentially, any liquid chromatographic separation mode can be used in conjunction with microdialysis, though the most common are reversed-phase (Cia and CK) materials. Separated compounds are detected most often using electrochemical, fluorescence, and UV detectors. However, some precautions related to the specific characteristics of microdialysis samples should be kept in mind. First, an average total sample volume is usually about 10 ¿iL, and the concentrations of analytes are very low, often close to the limits of detection of most instruments. Therefore, miniaturization of chromatographic systems will be important for continued advances in microdialysis. Microdialysis samples are free from large molecules, lipids, and other anomalies and can therefore be injected directly onto the chromatographic column. Microdialysis sampling is rapid—some 50 samples per day can easily be generated. It is important to optimize the separations for the fastest possible analysis time, often sacrificing the complete resolution of uninterested peaks. The use of automated

Figure 6,4 The starting setup for the clinical use of microdialysis in human adipose tissue consists of a portable, battery-driven syringe micropump (CMA 106) and a flexible microdialysis catheter (CMA 60). The catheter (.4) is introduced into the tissue by means of a special introducer (8). The needle has a longitudinal channel opening on its side, which allows its removal from the tissue and disconnection from the microdialysis catheter. The sample is collected into a special microvial (C).

Figure 6,4 The starting setup for the clinical use of microdialysis in human adipose tissue consists of a portable, battery-driven syringe micropump (CMA 106) and a flexible microdialysis catheter (CMA 60). The catheter (.4) is introduced into the tissue by means of a special introducer (8). The needle has a longitudinal channel opening on its side, which allows its removal from the tissue and disconnection from the microdialysis catheter. The sample is collected into a special microvial (C).

systems including autosamplers and data acquisition is strongly recommended. A refrigerated autosampler (CMA 200), injecting volumes from 0.1 ¿iL with almost no sample loss, has been built specially for microdialysis samples.

6.2.3 Performing a Microdialysis Experiment on a Rat

Besides the basic apparatus for microdialysis perfusions, fraction collection, and HPLC analysis, several additional instruments and devices are needed, depending on where the microdialysis probe is to be implanted. The most complicated instrumental setup is probably that required for brain dialysis. A stereotaxic instrument and a stereomicroscope are necessary for precise positioning of microdialysis cannulae into various brain structures. Inhalation anesthesia is preferable and more convenient than injections. However, this type of anesthesia calls for additional equipment, such as air lines, valves, and mixing chamber for halothane or other anesthetic gases, as well as good ventilation of the operation theater.

Once a laboratory animal such as a rat has been anesthetized, it can be fixed into the stereotaxic frame following procedures described in brain stereotaxic atlases (Paxinos and Watson, 1982). The points for fixation are the interaural line and the bottom edge of the upper jaw behind the first incisors. The skull must be exposed to locate the brain coordinates corresponding to the atlas nomenclature. Typically, the bregma (intersection of coronal and sagittal sutures) is chosen as zero "x,y" coordinates. A fine trephine drill should be used to make a hole of some 1 to 2 mm, and all the bone fragments must be carefully removed to avoid damaging the membrane during probe implantation. The surface of the dura mater is usually taken as zero for the "z" coordinate. After the zero coordinates have been marked, a sharp needle is used to cut the dura mater, and the microdialysis probe can be inserted. Some 1 to 3 hours is recommended for stabilization of the outflow of the measured substances. Then three to five basal level fractions are collected before particular stimuli (drugs, ischemia, etc.) are introduced.

There is also the possibility of performing experiments on conscious, free-moving animals. In this case, only the guide cannula is implanted into the anaesthetized animal. Following postoperative recovery (1-7 days), the probe can be inserted directly into the brain through the guide cannula without any need for anesthesia. Chronic microdialysis experiments can be run for up to 3 to 4 days after implantation (Osborne et al., 1991) and still give physiologically relevant data. After 4 to 7 days, a number of tissue reactions such as gliosis and accumulation of polymorphonuclear leukocytes (Benveniste and Diemer, 1987) cause severe alterations in both metabolism and diffusion rate of compounds in the vicinity of the microdialysis probe (Westerink and Tuinte, 1986).

6.2.4 Performing a Microdialysis Experiment on a Human

Although as yet there is no generally approved probe (except in Sweden) for clinicial microdialysis in humans, a number of papers have reported studies on human brain (Hillered et al., 1990; Hillered and Persson, 1991; During, 1991; During et al., 1994), heart (Kannergren et al., 1994), muscle (Rosdahl et al., 1993), skin (Petersen et al., 1992, 1994; Anderson et al., 1994), and adipose tissue (Jansson et al., 1988; Bolinder et al., 1989; Hagström et al., 1990; Meyerhoff et al., 1994). One of the most attractive potential applications is monitoring the subcutaneous glucose levels of diabetic patients (Bolinder et al., 1992, 1993; Pfeiffer, 1994; Sternberg et al., 1994) or newborn children (Korf et al., 1993; DeBoer et al., 1994).

For this application, a sterile microdialysis catheter is implanted percutane-ously into the subcutaneous adipose tissue in the periumbilical region using a special stainless steel guide cannula (Rosdahl et al., 1993). The membrane length of 2 to 3 cm and flow rates of 0.1 to 0.5 /xL/min should guarantee sufficiently long dialysis time to achieve 100% recovery. Another approach is to use a flat dialysis probe for transcutaneous applications (DeBoer et al., 1993; Korf et al., 1993). Here the skin of newborn babies is first partially removed by stripping with medical tape. Then a microdialysis probe is placed directly onto the exposed skin, usually on the abdomen lateral to the umbilicus (DeBoer et al., 1994).

6.3 APPLICATIONS OF MICRODIALYSIS/HPLC IN ENZYMATIC ANALYSIS

6.3.1 Body Fluids Sampled by Microdialysis

The main target for microdialysis implantations is the ECF of various organs, predominantly the brain. The main objective of these studies is to find chemical correlations, as represented by measured neurotransmitter levels, to pharmaceutical, behavioral, pathological, or other stimuli. However, because of its small size a microdialysis probe allows sampling of fluids from other locations without first removing the fluid. Body fluids other than ECF to which the microdialysis technique has been applied are considered in Sections 6.3.1.1 to 6.3.1.6.

6.3.1.1 Blood Microdialysis in blood can be performed both acutely, using rigid probes and guide cannulae, and chronically by implanting flexible probes with a removable guide-introducer needle. The most common site of implantation is a jugular vein (Hurd et al., 1988; Ekstrom et al., 1994) or the vena cava (Dubey et al., 1989). The method is attractive for the studies of protein binding (Saisho and Umeda, 1991; SjOberg et al., 1992; Nakashima et al., 1994), and pharmacokinetic and pharmacodynamic tests (St&hle, 1991; St&hle et al., 1993; Wong et al., 1993, Delange et al., 1994). Some advantages are the sampling of free unbound fractions and the possibility of long-term experiments without removing blood or affecting the animal's physiology. Microdi-

alysis is especially useful for bioavailability estimations, since a direct kinetic profile is produced (Wong et al., 1992; Delange et al., 1995).

6.3.1.2 Cerebrospinal Fluid (CSF) Microdialysis allows sampling of CSF (Golden et al., 1993; Malhotra et al., 1994; Togashi et al., 1994) without removal of the fluid and without altering intracranial pressure. CSF can be sampled from the lateral ventricles (Becker et al., 1988) or from the lumbal spinal cord area (Marsala et al., 1995), but most often CFS from the cisterna magna is tested (Knuckey et al., 1991; Matos et al., 1992). To date all studies have been performed in the rat.

6.3.1.3 Vitreous Humor A number of microdialysis studies have been carried on the vitreous fluid of a rabbit eye (Gunnarson et al., 1987; Waga et al., 1991) to study the penetration into this compartment of drugs, including antibiotics (Ben-Nun et al., 1988b) and cytostatics and corticosteroids (Waga and Ehinger, 1995). Polyamide membranes were found to be more suitable than polycarbonate for lipophilic compounds because of their lower carryover and memory effect for these drugs (Waga and Ehinger, 1995). The pathology of the eye was studied using models of experimentally induced ischemia of the retina (Ben-Nun et al., 1988a; Louzadajunior et al., 1992) and laser coagulation (Stempels et al., 1994).

6.3.1.4 Synovial Fluid The compartment of a joint space in the rat knee is filled with the synovial fluid. It has been shown that a microdialysis probe with the 0.5 mm membrane can be used for kinetics and distribution studies of drugs such as bis(5-amidino-2-benzimidazolyl)methane in experimentally induced arthritis (St. Claire and Brouwer, 1992).

6.3.1.5 Perilymph The cochlea of the inner ear contains two compartments: the scala tympani, filled with perilymphatic fluid, and the scala media, filled with endolymphatic fluid. Microdialysis has been performed in the scala tympani of a guinea pig (Laureil et al., 1994). The aim of this study was to measure cisplatin accumulation in this compartment, which has estimated volume of about 15 juJL.

6.3.1.6 Bile Microdialysis in the bile duct was performed using a specially constructed shunt probe (Scott and Lunte, 1993). The probe resembles in principle a classical dialysis cartridge with only a single dialysis tube inserted in the outer tubing. The ends of the dialysis tube are inserted into the vessel or bile duct, while the outer tubing is perfused with a Ringer's solution containing sodium taurocholate. Dialysates are collected, and various compounds such as drugs and their metabolites can be determined by HPLC.

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