Probes, dialysis

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. 

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 4.8 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 )...

Chaires JB (2005) Structural Selectivity of Drug-Nucleic Acid Interactions Probed by Competition Dialysis. 253 33-53 Cherkinsky M, see Braverman S (2007) 275 67-101... 

The first pH sensor was developed at NIH (Bethesda, Maryland) and made use of phenol red as acid-base indicator, covalently bound to polyacrylamide microspheres10 such microspheres are contained inside a cellulose dialysis tubing (internal diameter 0.3 mm) connected to a 250 pm plastic fibre (Figure 2). The probe was inserted into either the tissue or the... 

Purify the thiolated oligonucleotide from excess DTT by dialysis or gel filtration using 50mM sodium phosphate, ImM EDTA, pH 7.2. The modified probe should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this chapter) for nick-translation modification of DNA probes. Alternatively, dialysis, gel filtration, or w-butanol extraction may be used to remove excess reagents. 

Purify the biotinylated DNA probe by ethanol precipitation, gel filtration, w-butanol extraction, or dialysis as discussed in previous sections. 

The encapsulation of small guest molecules was achieved by constructing the dendrimer shell in the presence of the guest molecules followed by extensive dialysis to remove free guest molecules in solution [11]. A variety of probe molecules were applied, including Rose Bengal, 7,7,8,8,-tetracyano-quino-dimethane (TCNQ) and 3-carboxy-PROXYL. UV-Vis, fluorescence and EPR... 

Fluorescence resonance energy transfer has also been used for ionic strength measurements.(95) Fluorescein labeled dextran (donor) and polyethyleneimine-Texas Red (acceptor) were placed behind a dialysis membrane. The polymer association is ionic strength dependent and the ratio of intensities (F o/Fw) was used as the measured parameter. Since both the donor and acceptor are fluorescent, this kind of sensor may allow expand the sensitive ionic strength range by shifts in observation wavelength, as was discussed for pH probe Carboxy SNAFL-2 (see Section 10.3). 

The technique consists of a microdialysis probe, a thin hollow tube made of a semi-permeable membrane usually around 200-500 /xm in diameter, which is implanted into the skin and perfused with a receiver solution that recovers the unbound permeant from the local area. In principle, the driving force of dialysis is the concentration gradient existing between two compartments separated by a semi-permeable membrane. For skin under in vivo conditions, these compartments represent the dermal or subcutaneous extracellular fluid (depending on the probe position) and an artificial physiological solution inside the probe [36-38],... 

In vivo microdialysis is based on the principle of dialysis, the process whereby concentration gradients drive the movement of small molecules and water through a semipermeable membrane. In vivo microdialysis involves the insertion of a small semipermeable membrane into a specific region of a living animal, such as the brain. The assembly that contains this semipermeable membrane is called a probe, which is composed of an inlet and an outlet compartment surrounded by a semipermeable membrane (see O Figure 9-1). Using a microinfusion pump set at a low flow rate (0.2-3 /rL/min), an aqueous solution known as the perfusate is pumped into the inlet compartment of the microdialysis probe. Ideally, the... 

Two in vivo microdialysis techniques are employed in the clinical setting open and closed, which refer to the degree of exposure of the brain (Kanthan and Shuaib, 1995). In the open technique, a craniotomy is performed to expose the brain and the microdialysis probe is then implanted into the brain region of interest. In the closed technique, a burr hole is made in the skull and a modified probe is then inserted into the brain via the burr hole. The closed technique permits multiple insertions of the dialysis probe into the brain tissue and consequently minimizes problems associated with prolonged probe placement, such as edema and gliosis (Kochs, 1997). Therefore, the closed technique allows for continuous but intermittent sampling and has lower risks of tissue damage and infection than the open method (Kanthan and Shuaib, 1995). 

Responses in the dopamine system are more complex (see chapter by Balfour, this volume). Repeated nicotine injections resulted in enhanced extracellular DA levels in the NAc (Benwell and Balfour 1992, 1997), but not in the striatum (Benwell and Balfour 1997). Analysis of the precise placement of dialysis probes has revealed differential responses to drugs of abuse, including nicotine, between the NAc core (ventral striatum) and shell (Di Chiara 2002 Balfour 2004 Wonnacott et al. 2005 see chapter by Balfour, this volume). Moreover, the sensitised neurotransmitter responses observed in the hippocampus and NAc were markedly attenuated if rats received a constant infusion of a low level of nicotine (Benwell and Balfour 1997). Thus, transient peaks of nicotine appear capable of sensitising some brain pathways with respect to catecholamine release, but the responses may be mitigated by lower sustained plasma concentrations, possibly due to desensitisation. The extent that presynaptic nAChRs contribute to this process in vivo is unclear presynaptic a7 nAChRs on glutamatergic afferents to the VTA merit attention as potential mediators of sensitisation (see Sect. 2.2.2). 

Bacterial electrodes [11, 31, 33, 46, 48, 49, 60] In this type of electrode, a suspension of suitable bacteria is placed between the sensor proper and a dialysis membrane that prevents passage of high-molecular substances (see fig. 8.3). The sensor is usually a gas probe. In the simple types of bacterial electrode, the determinand is converted by a suitable strain of bacteria into a product sensed by the gas probe. Thus it is possible to determine arginine [46], glutamine [48],/.-aspartic acid [31],/.-histidine [60] and nitrate [33]. Hybrid bacterial - enzyme electrodes contain both a bacterial strain and a suitable enzyme. For example, an extract from ivingas Neurospora chossa can be used as a source of NAD nucleosidase and an Escherichia coli culture as a source of nicotinamide deaminase, so that the electrode responds to NAD [49] as a result of the series of reactions... 

Dialysis is the process in which small molecules diffuse across a semipermeable membrane that has pore sizes large enough to pass small molecules but not large ones. A microdialysis probe has a semipermeable membrane attached to the shaft of a hypodermic needle, which can be inserted into an animal. Fluid is pumped through the probe from the inlet to the outlet. Small molecules from the animal diffuse into the probe and are rapidly transported to the outlet. Fluid exiting the probe (dialysate) can be analyzed by liquid chromatography. 

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