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Solder

Joint

Insulated

Tin Wire

Polystyrene

Membraned Gold

Wire

Epoxy

Resin

BaU

Oxygen tension electrode, wire which acted as a small "spring

to follow the pulsatile movements of the brain cortex.

showing bends in the tin " to enable the electrode

The electrodes consisted of a short length of 125 micron diam e t e r pure gold wire (Goodfellow Metals) soldered to an

insulated tin wire of 500 micron diameter (RS Components). A low temperature soldering bit (260°C) and solder (60% tin 40% lead, 183°C melting point) was used to prevent vaporisation of the fine gold wire. Care was taken during the soldering process to produce a very small junction of the two wires wi t h the distance from the insulation to pure gold being no more than one millimetre. Epoxy resin (Araldite Rapide; Ciba Geigy) was applied to this junction and moulded into a small ball. By leaving the resin to cure slightly before application, it was possible to prevent it spreading over the gold wire. After drying overnight, the exposed gold wire was trimmed back to 1 .5mm using a scalpel blade. The cut was made ob liquely so as to provide a sharp point for smoother insertion into the brain. A m i c roscope was required for the cutting process, and at this point a check of the gold surface was made to ensure cleanliness and straightness, and the epoxy was inspected to ensure complete insulation of the j u n c t i o n .

The d i ffusion membrane was produced using a 10% w/v solution of polystyrene (BDH) in CCI* (AnalaR, B D H ) (Naylor and Evans, 1963). This was stored in a tightly capped vessel with minimal air space to prevent CCI4 evaporation from altering

the solution concentration. The solution could be stored in this way for several months. On the day prior to an experiment, the electrodes were dipped quickly into the solution up to the epoxy ball and then withd r a w n at a slow even pace. This was conducted smoothly so as to prevent drips of the solution forming on the wire and creating an uneven diff u s i o n barrier. After drying for ten minutes in the upright position, the process was repeated. The electrodes were left to dry for 6 to 8 hours, and then immersed in

E lec t r o n i c Circuitry

A p o l a r isation voltage of -800mv was required to reduce o x ygen at the surface of the electrode. However, the use of hydrogen clearance for the measurement of blood flow required an electrode polarisation of +400mv. To combine the two recording techniques in the same animal, the following circuitry was developed (figure 2.2.b). Figure 2.3.b is a schematic representation of the polar i s i n g voltages achieved by this circuitry.

The reference electrode polarised the electrically floating animal or recording m e dium to -400mV. W hen the oxygen or blood flow electrodes were placed in the animal, they became polarised to +400mV with respect to the reference voltage. In o r der to polarise the oxygen electrodes to -800mv in the +400mV recording environment, each electrode was fitted with a floating nickel/cadmium battery to polarise the electrode to -1.2V. With respect to the reference voltage of -400mV the electrode surface charge became -800mV, the o p t imum for ox ygen reduction (Fatt, 1976).

The amplifier system operated in the same way as the blood flow system with simple balance and gain controls, except that the response time of the circuit to a square wave input was reduced to approximately 1 .5 s e c o n d s .

■0.4

1.2

REF

OUT

OUT

+0.4

Figure 2.3.b

Schematic diagram of the polarisation voltages induced by the electronic circuitry used to measure blood flow and oxygen tension.

In V i t r o Calibration

Cali b r a t i o n of the oxygen electrodes was p e rformed in a system that mimicked the electronic and physiological environment of the animal model. A glass vessel was designed and m a n u f actured (Radleys) to provide a constant temperature and controlled distribution of c a l ibrating gases (Figures 2.3.c). The water jacket was fed from a c i r c ulating water b ath (Julabo) to maintain a temperature of 37°C. The flat base of the vessel allowed it to stand on a m agnetic stirrer, and a large 'flea' was used to swirl the gas that bubbled from the scintillated base. The gases were prewarmed by d e l ivering them though a tube that passed inside the water jacket. The perspex lid was a good fit on the flat edges of the vessel and reduced the interference of the atmosphere on the calibrating solution. The opening for the electrodes was small and the flow of gas from the chamber prevented the entry of the surrounding air.

The calibrating gases were ready m ixed at about 6 , 3 and zero

p ercent oxygen in nitrogen (BOC Special Gases). All the gases w ere supplied with calibration certificates for accurate d e termination of the oxygen tension they produced. The gases w ere delivered to the calibration chamber though a manifold of taps to allow rapid switching from one gas to another.

The electrodes were held in the chamber by securing them to glass microscope slides and then lowering the slide through the opening in the perspex top. The chamber was filled with 400ml of ISOmM sodium chloride. Anot h e r small hole in the lid allowed access of a polarising s i lver/silver chloride plate electrode (Clark Electromedical). Test i n g of the electrodes was carried out after soldering m u l t i s t r a n d e d wires to the stripped ends of the tinned wire, allowing connection to a total of eight amplifier units.

Before the electrodes were connected to the amplifiers, the balance of the circuit was adjusted to set the output to

PERSPEX TOP. WATER IN. OPENING CHAMBER OF SALINE