Microdialysis

PURPOSE AND RATIONALE Distribution in vivo could be studied by microdialysis. Microdialysis is an in vivo technique that permits measurement of unbound drug or metabolite concentrations in extracellular fluid of specific tissue location. The unbound drag concentrations have been shown to be responsible for the pharmacological effects. The basic principle is to mimic the function of a capillary blood vessel by perfusing a thin dialysis tube implanted into the tissue with a physiological liquid (Ungerstedt). 

Quantitative values on the unbound drug or metabolite concentration-time profile in different tissues or sub-regions of tissues are determined. 

Microdialysis has a number of advantages concentration profiles of drug could be obtained without fluid loss from freely moving individual subjects in specific sub regions of tissues. 

Microdialysis has also a number of disadvantages the probe could elicit tissue trauma after implantation, the determination of in vivo recovery is time-consuming and sensitive analytical methods are needed due to the diluting effect (De Lange). 

New probes (Evrard), methods for analysis and for recovery are developed. The optimal conditions for composition of the perfusion solution, the flow-rate, the post-surgery interval are searched. 

The principle of dialysis is based on the free movement of small molecules through a semipermeable membrane (thickness 9-30 pm), whereas larger molecules (e.g., proteins) are not able to cross. Small molecules move through diffusion from a high-concentration zone to a low-concentration zone. Scaling down this process to small hollow fiber membranes is referred to as microdialysis. This sample preparation technique can be used to isolate compounds from tissue or other complex samples. In contrast to most other sample preparation techniques used for biological samples, only the unbound free fradion of the analyte is isolated in this method. 

This is of particular interest for determination of drugs. Most drugs bind to proteins to a certain extent (up to 99%) and only the unbound fraction is biologically active. In such cases, microdialysis gives an estimate of realistic free concentration. Factors such as flow rate of the perfusate, diameter and length of the membrane, molecular mass cutoff, and membrane composition have influence on microdialysis. 

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The microdialysis sampling process which allows the monitoring of small molecules in circulation within an animal, is an example. An artificial capillary is placed in the tissue region of interest, and a sample is coUected via dialysis. In the case of a laboratory animal such as a rat, a probe is placed in the jugular vein under anesthesia. Elow rates ate of the order of 1 p.L/min. 

T. Foi nstedt, A.-M. Hesselgren and M. Johansson, Chiral assay of atenolol present in microdialysis and plasma samples of rats using chiral CBH as stationaiy phase . Chirality 9 329-334 (1997). 

Howard SG, Feigenbaum JJ Effect of gamma-hydroxybutyrate on central dopamine release in vivo a microdialysis study in awake and anesthetized animals. Biochem Pharmacol 33 103-110, 1997... 

Ahn JK, Koh EM, Cha HS, Lee YS, Kim J, Bae EK (2008) Role of hypoxia-inducible factor-lalphain hypoxia-induced expressions of IL-8, MMP-1 and MMP-3 in rheumatoid fibroblast-tike synoviocytes. Rheumatology (Oxford) 47(6) 834-839 Ao X, Wang X, Lennartz MR, Loegering DJ, Stenken JA (2006) Multiplexed cytokine detection in microUter microdialysis samples obtained from activated cultured macrophages. J Pharm Biomed Anal 40(4) 915-921... 

Newer techniques include monitors capable of performing microdialysis, or measuring brain oxygenation and lactate, which may be useful in monitoring penumbral tissue adjacent to a large area of infarction. No randomized studies have been performed to clearly document their impact on patient outcomes to date. 

Johnston AJ, Gupta AK. Advanced monitoring in the neurology intensive care unit microdialysis. Curr Opin Crit Care 2002 8(2) 121-127. 

Figure 4.6 The tip of a microdialysis probe, expanded to show dialysis tubing around a steel cannula through the base of which fluid can flow out and then up and over the membrane. The length of membrane below the probe support can be altered (1-10 mm) to suit the size of the animal and the brain area being studied. Flow rates are normally below 2 pl/min...

Figure 4,7 The effect of perfusion of the microdialysis probe with a medium containing a depolarising (80 mM) concentration of K" ", or Ca +-free medium, for the periods indicated by the bars. The graph shows efflux of noradrenaline in the frontal cortex of anaesthetised rats. Increasing the concentration of K" " in the medium infused via the probe increases noradrenaline efflux whereas removing Ca reduces it...

Figure 4.8 Noradrenaline concentration in dialysis samples from probes implanted in the rat frontal cortex. Spontaneous efflux of noradrenaline is stable throughout a 4h sampling period ( extended basals ) but is increased markedly when either the noradrenaline reuptake inhibitor, desipramine (5 pM), or the a2-adrenoceptor antagonist, atipamezole (0.5 pM), is infused into the extracellular fluid via the microdialysis probe ( retrodialysis )...

Flentge, F, Venema, K, Koch, T and Korf, J (1997) An enz5mie-reactor for electrochemical monitoring of choline and acetylcholine. Applications in high-performance liquid chromatography, brain tissue, microdialysis and cerebral fluid. Annal. Biochem. 204 305-311. 

Even if this turns out to be the case, it is likely that noradrenergic neurons in different brain regions make different contributions to this process. This complication is suggested by the results of a recent microdialysis study in which release of noradrenaline in response to the sound of a buzzer alone was provoked after repeated... 

Figure 8.11 Noradrenaline efflux, measured by microdialysis, in the rat frontal cortex and hypothalamus, (a) Repeated exposure to a tone, alone, has no effect on noradrenaline efflux in either brain region, (b) After repeated pairing of the tone with transfer of the rat to a brightly lit (aversive) arena, the sound of the tone alone triggers a significant ( P<0.05, cf last basal sample) increase in noradrenaline efflux in the frontal cortex, but not the hypothalamus. (Based on a figure from McQuade and Stanford 2000)...

McQuade, R and Stanford, SC (2000) A microdialysis study of the noradrenergic response in rat frontal cortex and hypothalamus to a conditioned cue for aversive, naturalistic environmental stimuli. Psychopharmacology 148 201-208. 

The comparatively straightforward link between 5-HT and its primary metabolite, 5-HIAA, encouraged many researchers to use changes in the ratio of tissue concentrations of 5-HIAA and 5-HT as an index of the rate of release of 5-HT ex vivo. However, it has been clear for some time that the majority of 5-HT is metabolised in the cytoplasm by MAO before it is released from 5-HT nerve terminals. Consequently, the reliability of the 5-HIAA 5-HT ratio as an index of transmitter release is rather dubious, although it could be used as an acceptable measure of MAO activity. In any case, the development of in vivo microdialysis means that changes in the concentration of extracellular 5-HT can now be monitored directly which, under drug-free conditions, provides a far more reliable indication of any changes in the rate of release of 5-HT. 

To some extent, this proposal is supported by microdialysis studies of changes in 5-HT efflux in the terminal fields of 5-HT neurons. For instance, increased 5-HT efilux in the striatum, induced by immobilisation of rats, occurs only during the period of increased motor activity that follows the animals release (Takahashi et al. 1998). A single swim stress also fails to increase 5-HT efflux in the medial prefrontal cortex of rats. 

Obviously, regulation of food intake depends on many neurotransmitters and hormones but this final section will outline the role played by central 5-HT transmission in this process. It had been the belief for some time that increased 5-HT transmission in the brain reduces food intake (Blundell 1977) and this certainly explains the satiety in rats that follows infusion of 5-HT into the paraventricular nucleus (PVN) of the hypothalamus. However, recent studies using microdialysis have found that 5-HT efflux in the lateral hypothalamus is itself increased by food intake, suggesting the existence of a feedback control system. In fact, because the increase in 5-HT efflux is greater in genetically obese rats than in their lean counterparts, it has been proposed that there is a deficiency in the 5-HT inhibition of food intake in obesity. 

An important distinction between the effects of sibutramine and i/-fenfluramine is highlighted by microdialysis studies (Heal et al. 1998). These show that the rate of increase in 5-HT efflux in the region of the PVN, after administration of sibutramine, is slow, progressive and long-lasting. This is because it relies on the accumulation of extracellular 5-HT following the inhibition of its reuptake after impulse-dependent release. This time-course contrasts with the rapid and transient increase in 5-HT efflux which results from the fenfluramine type of impulse-independent release from nerve terminals. In fact, this rapid increase in 5-HT release is thought to underlie the serious adverse side-effects of (i-fenfluramine that have led to its withdrawal from the clinic. 

Petty, F, Jordan, S, Kramer, GL, Zukas, PK and Wu, J (1997) Benzodiazepine prevention of swim-stress induced sensitization of cortical biogenic amines an in vivo microdialysis study. Neurochem. Res. 22 1101-1104. 

Rouch, C, Nicolaidis, S and Orosco, M (1999) Determination, using microdialysis, of hypothalamic serotonin variations in response to different macronutrients. Physiol. Behav. 65 653-657. 

ATP certainly fulfils the criteria for a NT. It is mostly synthesised by mitochondrial oxidative phosphorylation using glucose taken up by the nerve terminal. Much of that ATP is, of course, required to help maintain Na+/K+ ATPase activity and the resting membrane potential as well as a Ca +ATPase, protein kinases and the vesicular binding and release of various NTs. But that leaves some for release as a NT. This has been shown in many peripheral tissues and organs with sympathetic and parasympathetic innervation as well as in brain slices, synaptosomes and from in vivo studies with microdialysis and the cortical cup. There is also evidence that in sympathetically innervated tissue some extracellular ATP originates from the activated postsynaptic cell. While most of the released ATP comes from vesicles containing other NTs, some... 

Histamine is synthesised by decarboxylation of histidine, its amino-acid precursor, by the specific enzyme histidine decarboxylase, which like glutaminic acid decarboxylase requires pyridoxal phosphate as co-factor. Histidine is a poor substrate for the L-amino-acid decarboxylase responsible for DA and NA synthesis. The synthesis of histamine in the brain can be increased by the administration of histidine, so its decarboxylase is presumably not saturated normally, but it can be inhibited by a fluoromethylhistidine. No high-affinity neuronal uptake has been demonstrated for histamine although after initial metabolism by histamine A-methyl transferase to 3-methylhistamine, it is deaminated by intraneuronal MAOb to 3-methylimidazole acetic acid (Fig. 13.4). A Ca +-dependent KCl-induced release of histamine has been demonstrated by microdialysis in the rat hypothalamus (Russell et al. 1990) but its overflow in some areas, such as the striatum, is neither increased by KCl nor reduced by tetradotoxin and probably comes from mast cells. 

The ability of the striatum to apparently function normally until it has lost much of its DA can be ascribed in part to denervation supersensitivity, the degeneration of the DA input resulting in an increase in postsynaptic DA receptors and partly to the remaining neurons producing more DA. This is supported by measurements in humans which show that the HVA DA ratio, a measure of DA turnover, is much greater in Parkinsonism patients and by microdialysis in rats with 6-OHDA lesions of the nigrostriatal tract, when the reduction in perfusate (released) DA is very much less than that of neuronal (stored) DA. 

There is certainly evidence that whereas typical neuroleptics are equally active in mesolimbic/cortical areas as well as the striatum, the atypical drugs are much less effective in the latter. This has been shown by (1) increased DA turnover through DOPAC and HVA production in vitro, (2) augmented DA and DOPAC release by microdialysis in vivo and (3) increased c-fos- ike, expression. 

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