In-vitro superfusion system (Cortical Wedge technique)

In-vitro superfusion systemIn-vitro superfusion system

The in-vitro two-chamber superfusion system is one of the most important members of the MDE GmbH nerve tissue examination equipment. The nerve tissues are spatially separated in the split chamber, but functionally they remain connected.

The two-chamber brain slice perfusion system is the unique combination of the wedge preparation technique, the in-vitro superfusion system and the radioactive neurotransmitter release technique. Its purpose is to evoke neurotransmitter release and biopotential responses in one part of the nerve tissue by selective stimulation of the other part, and to simultaneously measure the two responses.

System

Scheme of the systemScheme of the system

Main compnents

  • Central unit with measuring chambers and pre-heaters,
  • Biopotential amplifier and electrostimulator,
  • Heat stabilizer and buffer pump.

 

 

Central unitCentral unit

The components of the central unit are placed on a specially designed stand in the following order, top to bottom:

  • Incubation chambers with carbogen controllers,
  • Measuring chambers with bubble trap, divider disks, and stimulating and measuring electrodes,
  • Pre-heater.

 

Incubation chamber with carbogen controller

Incubation chambers with carbogen controllersIncubation chambers with carbogen controllers

The double-walled, 10 ml capacity chamber is connected to the thermo stabilizing unit through the liquid circulating in the jacket. The chamber is to keep the tissue slices prepared for the measurement alive, carbogenized and at appropriate temperature, until the measurement starts. Continuous supply and appropriate condition of the gas necessary for incubation is provided by the buffer vessel, and the fine adjuster and atomizer unit.

The unit makes the process of measurement easier and faster.

 

Measuring unit

Measuring unitMeasuring unit

Measuring chamberMeasuring chamber

The physiological conditions required to keep the tissue physiologically stable in the measuring chamber are ensured by the heat-stabilized physiological solution (buffer) running through the pre-heater. The optimal fluid level of the chambers is secured by continuous in-and outflow via a peristaltic pump.

The two half-chambers are separated by a specially shaped spacer disk with silicone sealing.

Stimulating and measuring electrodes are found in both half-chambers, enabling bidirectional stimulation and measurement. Two measuring stubs, suitable to accept thermosensors, are provided for temperature adjustment and continuous monitoring of the chamber liquid (optional accessories, built in only on request).

Measuring chambers

Drawing of the measuring chamberDrawing of the measuring chamber

Measuring chamberMeasuring chamber

Measuring chamberMeasuring chamber

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 tissue can be positioned in the fluid space in such a way (e.g. for Cortical Wedge) that the cortical and the striatal parts are separated in the line of the corpus callosum. 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 two compartments are independently supplied with puffer by means of a peristaltic pump. The incoming liquid first enters the small chamber in front of the compartments. There, on the one hand, bubbles are removed from the heated, oxygenized liquid; on the other hand the periodic liquid waves generated by the peristaltic pump are reduced. The inlet and outlet bore is placed staggered, to obtain a cross flow of liquid in the chamber with smallest dead space possible.

 

Biopotential amplifier (EXT MW-04)

Biopotential amplifierBiopotential amplifier

The amplifier was developed for the potential changes sensed by the Ag/AgCl electrodes. The sensors pick up the potential changes to be measured through the liquid, and transfer them directly coupled to the AC/DC input amplifier. The amplifier has internal calibration and offset equalizer. The analog output of the amplifier is compatible with any standard AD converter, making it connectable to any chart software in the market.

Technical parameters  
 Input resistance 10 MOhm
Amplification x100, x1000
Zero offset ±10 mV, relative to the input
Test circuit 10 kOhm /250 mV (gain= x100)
Output resistance 100 Ohm
Maximal Output Current 4 mA
Weight 8 kgs with power supply

Accessories required for operation

 CWB-02 Circulation water bath

Circulating water bathCirculating water bath

 PRO-IM-02 Peristaltic pump

Peristaltic pumpPeristaltic pump

 FC 203 B Fraction collector

Fraction collectorFraction collector

EXP-ST-04 Four-channel non-isolated stimulator with MCU controlling unit

Square wave stimulatorSquare wave stimulator

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 modelSchizophrenia 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 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.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

You find details by clicking on the product code.

ISN-02 Four-channel* in-vitro superfusion systems (Cortical Wedge technique)

Code Name
  Stand with silicone tubing
  Two-chamber tissue bath with isolation component, Ag/AgCl electrodes and stimulating electrode (4pcs)
  Pre-heater unit (8 pcs)
  Organ incubation chamber (4 pcs)
  Gas vaporizer set
  Four-channel broadband AC amplifier
  Eight-channel persitaltic pump (2 pcs)
OPTIONAL  
EXP-ST-04 Four-channel non-isolated stimulator with MCU controlling unit
CWB-02 Circulating water bath (20 litres, 20 l/min delivery speed, ±0,1°C)

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

References

 Year  Author  Title  Source
2004 Bujdosó E. Role of recently discovered opioid neuropeptides in the regulation of Open-Field Behaviour and the Hypothalamic-Pituitary-Adrenal axis Department of Pathophysiology, Albert Szent-Györgyi Medical and Pharmaceutical Center, Faculty of Medicine, University of Szeged
2009 Világi I, Dobó E, Borbély S, Czégé D, Molnár E, Mihály A. Repeated 4-aminopyridine induced seizures diminish the efficacy of glutamatergic transmission in the neocortex Exp Neurol. 2009 Sep;219(1):136-45. doi: 10.1016/j.expneurol.2009.05.005. Epub 2009 May 13.

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

Media

Isolated nerve tissue sytems cat  Download the Isolated nerve tissue systems catalogue in pdf version!

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