In-vitro superfusion system (Cortical Wedge technique)

How can it help?

The dual-chamber in-vitro perfusion system was developed from the combination of two well-known techniques (wedge evoked potential measurement and neurotransmitter release). Its purpose is to simultaneously measure the evoked potentials and neurotransmitter release in one part of the examined neural tissue on selective stimulation (electrical or chemical) of the other part of the tissue. The technique allows the simultaneous or temporally separated electrical stimulation of the two tissue areas. Furthermore, the separated, but anatomically connected tissue areas can be perfused with different substances (independently of each other, but also simultaneously).

The development of the system and related experiments were conducted in the research laboratory of Servier/Egis (L. Hársing Zs. Jurányi et. al.). The application possibilities and advantages of the system are presented with the results of two experiment series.

Schizophrenia model

In examining the neurochemical background of schizophrenia, it is a well-known practice to remove the striatum from the brain and place it into a one-volume perfusion chamber, to measure [³H] dopamine release in the presence of various compounds. Two nerve paths make connection in the striatum, one originating from the cerebral cortex and the other from the substantia nigra. Decay of the latter one leads to Parkinson's disease, while reduced activity of the former plays a role in the development of schizophrenia. According to literature data, neurochemical experiments performed on striatal slices in a one-volume perfusion chamber often delivered sharply contrasting results. During the preparation of striatal slices, the the mentioned nerve paths are inevitable cut. The resulting preparation contains the endings of both paths, but not the initial nerve cells and the intact pathways themselves. To achieve appropriate results, brain slices are necessary which include the cerebral cortex, the intact pathways and the striatum itself (where the path ends).

Schizophrenia model

During the experiment the cortex is located in one half of the chamber while the striatum is located in the other half. The tissue connection between the two tissues (corpus callosum) is preserved but there is no communication between the two fluid compartments. A plexiglass divider plate can be inserted between the two halves of the open organ chamber. The inserted plate divides the chamber into two compartments, each with 3 ml volume. The brain slice can be positioned in fluid space in such a way that the cortical and the striatal parts are separated in the line of the corpus callosum, so that the cortical part will be in one compartment and the striatal part will be in the other. The separating wall and the inserted tissue are surrounded by high purity silicone grease, so no liquid exchange can take place between the two compartments. The balanced flow of the solution is provided by a peristaltic pump.

Electrical stimulation of the tissue is provided by the platinum electrodes built into the bottom of the compartments in form of space stimulation (the tissue is situated between the two electrodes). Ag/AgCl electrodes built into the sidewalls detect depolarization caused by nervous activity as a potential difference between the two chamber spaces.

Electrical stimulation of the cortical part of the slice significantly increaes the [3H] dopamine level in the effluent from the chamber containing the striatal part of the brain slice.

Electrical stimulation of the cortex depolarizes the dopaminergic axon terminals in the cortical part of the slice, and they release their [³H]dopamine content which appears in the effluent. Simultaneously, as a result of the electrical stimulation, the corticostriatal glutamatergic pathways are also activated. Endogenous glutamate released from the pathways ending in the striatum stimulates the [³H] dopamine release from the nigrostrial dopaminergic endings.

Since the cortical part of the slice is located between the stimulating electrodes, electrical stimulation has a direct effect on the tissue. In contrast to that, the [³H] dopamine release observed in the striatum occurs secondarily as an effect of cortical stimulation. With this combined wedge-release method it is possible to examine the neurotransmitter release occurring simultaneously but spatially separated in the origination and termination areas of projection systems, together with the depolarization during nervous activity.

Ischaemia, atherosclerosis model

Electric voltage difference between the cortex and the striatum during a transient, experimental cortical ischemia. The red arrow indicates the onset of cortical ischemia. As a result of ischemia a depolarizing shift with spikes riding on it develops in the cortical area. This indicates electrical activity in the cortical area, burst-like discharge of neuronal groups. The orange arrow indicates the termination of temporary ischemia (perfusion continued with oxygenated, normal Krebs solution). It can be seen that the increased cortical activity, observed during ischemia, decreases rapidly. The yellow arrow indicates 3-minute perfusion of Krebs’ solution containing 40 mM KCl to the striatal area. The solution with increased KCl content creates an excessive depolarization in the striatum. Perfusion with KCl is used to monitor the viability of the striatum.

In case of circulatory insufficiency (heart failure, cardiac ischemia, myocardial infarction) or stenosis of the arteries of the central nervous system (atherosclerosis) temporary or permanent hypoxia, ischemia develops in the brain tissue.

The experimental system has the advantage that, thanks to the dual-chamber design, the effect of hypoxia in a given area of the nervous system on another area, unaffected by the hypoxia, can be modeled. In one half of an appropriate slice cut from the nervous system, that is, in one half-chamber, hypoxia- or ischemia-like state can be created while in the other half normoxic, glucose-containing physiological solution can be perfused further on.Meanwhile, electrical and/or chemical stimulation can be used (even on both sides) and transmitter release and/or potential changes induced by the pathological conditions can be examined. 

The figure demonstrates how experimental ischemia in one half of the brain slice (perfused with glucose-free Krebs’ solution saturated with N₂/CO₂) causes a clearly measurable potential change in the cortex. Via the two Ag/AgCl electrodes, this will be measured as potential difference between the cortex and the striatum.

Advantages of the system

1. One of the outstanding benefits of the open perfusion system, in contrast to closed systems, is that the test compounds can be injected directly next to the tissue in the chamber.
2. The grease-gap method with the perfusion chamber of appropriate precision, is suitable for studying the release of radioactive neurotransmitters in complex brain slices.
3. The two sides of the preparation can be perfused independently, which allows simultaneous delivery of different pharmaceuticals to both sides of the tissue.
4. The two sides of the preparation can be stimulated electrically independently of each other, which allows examination of the operation and impact of projection pathways originating in one half of the tissue slice and ending in the other half. If necessary, both sides of the slice can be stimulated (either simultaneously or alternately).
5. By means of the built-in Ag/AgCl electrodes, the potential changes generated in the tissue slice can be studied, providing information about the influence of the applied substances on neuronal activity.

Available models

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*The system is also available in one- or two-channel models, please, contact us for further details!

References

Note: experiments were performed with different types of isolated nerve tissue systems.

Media

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