EP1309856A2

EP1309856A2 - Use of an electrode array

Info

Publication number: EP1309856A2
Application number: EP01919364A
Authority: EP
Inventors: Cornelia Leibrock; Thomas Müller; Hansjürgen VOLKMER
Original assignee: NMI Naturwissenschaftliches und Medizinisches Institut
Current assignee: NMI Naturwissenschaftliches und Medizinisches Institut
Priority date: 2000-03-01
Filing date: 2001-03-01
Publication date: 2003-05-14
Also published as: WO2001065251A2; US20030068613A1; WO2001065251A3

Abstract

The use of an electrode array is disclosed, for the examination of a regeneration brought about in biological material by an active substance, or a physical method.

Description

Using an electrode array

The present invention is concerned with systems for studying regeneration, synaptogenesis and synaptic stability in biological material, such as cells.

The invention is also related to injuries to the central nervous system (CNS) of adult mammals caused, for example, by trauma, ischemia or neurodegenerative diseases. These injuries result in serious and permanent functional deficits that require curative treatment. It is already known that, in contrast to the developing CNS or peripheral nerves, CNS axons lose their ability to regenerate after axotomy at a certain point in development. This loss is believed to result from the non-permessive environment of the CNS and from factors intrinsic to the adult CNS neurons.

Treatments that can lead to neural regeneration include the administration of active substances, such as pharmaceuticals, or the use of physical methods, such as electrostimulation.

So far, no reliable method is known with which the evaluation of such treatments, which can lead to neuronal regeneration, is made possible within the framework of a model system that correlates long-term records of physiological activity with morphological changes.

Li et al-, Enthorinal Axons Project to Dentate Gyrus in Organotypic Slice Co-culture, NEUROSCIENCE 1993, Volume 52, pages 799-813 describe an organotypic coculture of the entorhinal cortex and postnatal hippocampus, which prepares itself from its own entorhinal entrance has been. In this in vitro system, the authors show the formation of entorhinal projections in the cocultivated hippocampus in an immunohistochemical manner, for which the sections were stained and evaluated under a microscope. The authors also show a few electrophysiological data that were obtained with an excitation electrode made of tungsten and a measuring electrode made of glass. The measurement method described here is not suitable as a model system for long-term recordings.

The exclusivity of the connections between the entorhinal cortex and the dentate gyrus is shown by Li et al., Connectional Specification of Regenerating Entorhinal Projection Neuron Classes Cannot Be Overriden by Altered Target Availability in Postnatal Organotypic Slice Co-culture, EXPERIMENTAL NEUROLOGY, 1996, volume 142, pages 151- 160, at the anatomical level.

Baker et al., Chronic Blockade of Glutamate-mediated Bio-electric Activity in Long-term Organotypic Neocortical Explantε Differentially Effects Pyramidal / Non-pyramidal Dendritic Morphology, DEVELOPMENTAL BRAIN RESEARCH, 1997, Volume 104, pages 31-39 show regeneration in another organotypic preparation, namely in cortex-cortex co-cultures. Electrophysiological data were also recorded here using glass microelectrodes.

Nessler and Mass, Direct-current Electrical Stimulation of Tendon Healing in vitro, CLINICAL ORTHOPEDICS, 1987, volume 217, pages 303-312 report on the possibility of stimulating tendon regeneration in vitro by direct current.

In addition, Borgens, Electrically Mediated Regeneration and Guidance of Adult Mammalian Spinal Axons into Polymeric Channels, NEUROSCIENCE, 1999, Volume 91, pages 251-264 was able to show that an extracellular electric field promotes the regeneration of nerve fibers in the adult mammalian spinal cord. The state of the art described so far shows basic model systems that are suitable for investigating the regeneration in biological material; However, reliable long-term measurements of the physiological activity and their correlation with morphological changes cannot be carried out with the measuring method described in the prior art.

WO 00/79273 A2, which was only published after the priority date of the present application, describes a method in which hippocampus tissue is arranged on a microelectrode array, via which the nerve tissue is stimulated and the different response to different psychotropic drugs is measured.

Against this background, the present invention has for its object to provide a way to evaluate treatments that can lead to neural regeneration in a model system that corrects long-term records of physiological activity with morphological changes.

This object is achieved according to the invention by using an electrode array in order to investigate a regeneration promoted by active substances or physical processes in biological material, a microelectrode arrangement being preferably used as the electrode arrangement.

The inventors of the present application have recognized that, in this context, organotypic cultures and cocultures of tissue of the central nervous system can be used, in particular, for the formation and regeneration of connections to measure functionally between cells and to investigate the effect of pharmaceuticals or physical processes for activating cell functions.

In the context of this application, promoting regeneration is understood to mean both the effect and the support of regeneration.

The inventors have also recognized and demonstrated that generally electrode arrays, especially microelectrode arrays, e.g. Egert et al., A Novel Organotypic Long-term Culture of the Rat Hippocampus on Substrate Integrated Multielectrode Arrays, BRAIN RESEARCH PROTOCOLS, 1998, Volume 2, pages 229-242, are particularly well suited for long-term measurement in such systems ,

By combining organotypic cocultures with extracellular microelectrode recording technology, the development of new connections and their regenerative ability can be repeatedly monitored on a functional level in the same culture for days to weeks.

In this context, organotypic cultures represent a good alternative to animal experiments for dealing with questions in the field of regeneration. The coculture model of the dentate gyrus and entorhinal cortex offers a favorable starting point. It can be seen from the literature described in the introduction that this coculture model is well morphologically characterized in the literature, and the analogy to the in vivo situation has also been demonstrated. However, electrophysiological data from the literature are hardly available. bar, these data can now be provided by using the microelectrode array (hereinafter: MEA). It is also advantageous that there is a simple, monosynaptic connection with a clearly defined starting and ending point between the two tissues.

Between the dentate gyrus and the entorhinal cortex there is a perforant path, well described in the literature, which is partially represented by this monosynaptic connection.

The inventors of the present application have now recognized for the first time that the use of microelectrode arrays makes it possible to generate a previously experimentally interrupted perforant pathway both during the juvenile phase, where regeneration is still relatively easy in the central nervous system, and in the differentiated state to investigate. The use of MEA technology in connection with the organotypical culture mentioned as an example offers the possibility of electrophysiologically determining the activity over the entire area of a preparation repeatedly over a period of days and weeks. Electrostimulation at reproducibly always the same places with simultaneous derivation in the entire remaining coculture is also possible in order to prove a connection between the two explants.

The explants grow together for the first time in the juvenile phase. If a lesion is then placed through the newly formed connection after the coculture has reached a differentiated state, the regeneration can also be examined in an adult-like state. Such long-term monitoring of 17 derived cocultures on an MEA with electrodes spaced 200 μm apart and 30 μm in diameter showed that twelve of these cocultures had grown together again. Seven of these co-cultures that had grown together were lesioned, four of which were still vital after the lesion.

It could be shown in an experiment that when using FK506, an immunosuppressor, a coculture regenerated the perforant pathway.

This is not only a test for the regenerative potential of the differentiated neurons, but also the regenerative potential of pharmaceuticals and physical processes for activating cell functions, e.g. by functional electrical stimulation. This can be done, for example, by direct electrical stimulation or the application of an electrical or electromagnetic field, which can also be carried out using an MEA.

According to the invention, the pharmaceuticals or active substances are selected from the group: organic or chemical compounds and biologically active substances, such as peptides, proteins, nucleic acids, where the biological material can include both juvenile cells and cells in an adult-like state.

Through the use according to the invention, the regeneration of intercellular connections, the functional interconnection or re-interconnection between neurons, a time course of the resulting connections or re-connection can now Circuits as well as the regeneration of cells damaged with regard to their intercellular connections are examined.

According to the invention, the regeneration of cultures and cocultures can also be observed repeatedly over a longer period of time, preferably more than one week. It is also possible to investigate the reactions of receptor systems to the administration of active substances.

Against this background, the application also relates to a method for investigating the effects of pharmaceuticals on the regeneration potential of functional interconnections in cultures, in which an electrode array is preferably used as described above.

It is first necessary to pharmacologically check which receptor systems in coculture are important, for example, for the connection from the entorhinal cortex to the dentate gyrus. For this purpose, the effect of antagonists of the various potentially relevant receptors on the activity in co-cultures grown together must be tested.

It goes without saying that the features mentioned above and those yet to be explained below can be used not only in the combination indicated in each case, but also in other combinations or on their own without departing from the scope of the present invention.

Further advantages result from the following exemplary embodiments and in connection with the drawing, in which: 1 shows a schematic representation of the in vivo situation between the entorhinal cortex (ec) and the dentate gyrus (dg);

2 shows the preparation of cocultures on a microelectrode array;

Figure 3 shows electrical stimulation on cocultures as in Figure 2;

Figure 4 shows the restoration of functional compounds in a coculture as in Figure 2; and

5 shows the electrical stimulation of electrogenic cells in the cell cluster on an MEA.

Example 1: Measurements on a coculture

Figure 1 is a schematic drawing of the in vivo situation between the entorhinal cortex (ec) and the dentate gyrus (dg). Part of the perforant pathway is shown with pp, which is cut during the preparation on the dashed line. CA1 and CA3 denote fields of the hippocampus.

Sections of the entorhinal cortex and dentate gyrus, which were taken from 6 to 7-day-old Wistar rats or BalbC mice, were cultured on a microelectrode array of 60 substrate-integrated electrodes. The electrodes had a stood of 200 μm and a diameter of 30 μm. The stimulation of the sections and the recording of the electrophysiological activity was achieved by using an MCS amplifier and a data acquisition system (Multi Channel Systems, Germany), which allows the use of each contact as a stimulation or recording electrode without manipulation of the section allows. The data was recorded online from all 60 channels at a sampling frequency of 25 kHz / channel.

2 shows such a preparation of the cocultures, for which horizontal brain sections (425 μm) were used. The entorhinal cortex and the corresponding dentate gyrus were separated from the brain sections, which represent the origin and target tissue of part of the preferant pathway. The cuts that were made during the preparation are shown in dashed lines in FIG. 2a.

The two explants were then positioned similar to the in vivo situation on a microelectrode array with an 8x8 field of extracellular electrodes. 2b shows a coculture immediately after preparation.

Starting from the seventh day in vitro (DIV7), every three days both the total spontaneous activity of the cocultures and the response to extracellular electrical stimulation of layer II of the entorhinal cortex and the stratum granulums of the dentate gyrus were monitored. Additionally marked Dil staining (1, l-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine perchlorate; Honey and Hume, Fluorescent Carbocyanine Dyes Allow Living Neurons of Identified Original to be Studied in Long-term Cultures, J CELL. BIOL. 1986, volume 103, pages 171-187) of the dg retrograd the cells in layer II of the ec.

FIG. 3 shows the extracellular stimulation of a coculture of ec and dg, FIG. 3a indicating the morphology of the coculture after 7 days in vitro (DIV7). 3b shows the reaction on all MEA electrodes after electrical stimulation in layer II-III of the ec (80 μA, 150 μs).

The functional restoration of compounds in cocultivated explants from dg and ec juvenile rats or mice is shown in FIG. 4. After 7 days in vitro, it is possible to elicit activity in the dg via electrical stimulation in the ec (FIG. 4a), but not vice versa (FIG. 4b). This corresponds to the in vivo situation in the ec layer II cells in the dg project.

Out of 112 cultures examined, 85 showed this unidirectional connection, 20 a bilateral connection and 7 no connection at all.

It can be concluded from these results that, regardless of the loss of the intermediate subicular tissue, the axons of the entorhinal cortex form correct and functional connections with the dentate gyrus, which are similar to the perforant pathway known in vivo. It is unlikely that the observed linkages from nonspecific axonal sprout originate, because the dg-axons also show extensive growth from the explant, but do not establish any functional links with the cortex.

The linked co-culture thus established is now developing in vitro to an adult-like stage, whereupon the newly produced compounds are then damaged.

The regenerative potential of the differentiated neurons and the promotion, i.e. effecting or supporting regeneration, of pharmaceuticals or physical processes can then be tested at these lesions.

Example 2: Investigations at different stages of regeneration

neurite outgrowth:

Neuroproductive or regenerative substances that act directly on the output cells in layer II of the ec can enable the pathway that has already been established in vitro to be regenerated, which is normally not possible in the differentiated state.

There is an experimental possibility here to apply FK506, for example, into the culture medium after the lesion. It is important that the culture has reached a sufficient age to prevent the neurites from growing due to development. This is the case after DIV7 or earlier. goal Setting

The repulsive effect of semaphorin III mediated by the neuropilin I receptor most likely contributes to the correct, targeted connection of dg and ec.

Experimentally, it is possible to inhibit the signal cascade in question with common peptides and to prevent the semaphorin III effect. The peptides are added to the culture medium immediately after preparation, so that a lesion is not necessary.

Svnaptoqenese

It has been found that the interaction with intracellular proteins is essential for the correct arrangement of the receptors and other membrane proteins in the synapse. Many of these intracellular proteins share a common motif, the so-called PDZ binding site, with which they bind to the membrane proteins. The functional relevance of these interactions is still largely unknown, but it can be investigated using the measurement method described here.

Here too, the interaction of the receptors with the PDZ proteins can be inhibited by membrane-permeable PDZ antagonists, which can take place during or after the growth. This makes it possible to compare the situation before / after a cut (internal control). Example 3: Immunohistochemical and electrophysiological examination

Organotypic cortical cocultures (350-425 µm thick, from the Neocortex) were prepared from three to seven day old Wistar rats of each sex and maintained in vitro using the roller tube technique; see Gähwiler, Organotypic Monolayer Cultures of Nervous Tissue, J. NEURO-SCIENCE METHODS, 1981, Volume 4, pages 329-342. The cocultures were grown for up to 35 days either on glass coverslips or on microelectrode arrays in medium containing 50% basic Eagle medium, 25% Hanks buffered saline, 25% horse serum, 33 mM D-glucose and 1 mM L-glutamine , The medium was changed twice a week, and the coculture explants were placed with different orientations and distances (0 to 500 μm) to one another.

The explants cultured on glass coverslips were fixed in buffered 4% paraformaldehyde for two hours, washed, treated in buffered 0.1% Triton X-100, washed again and in 1% bovine serum albumin (BSA) and 0.1% Goat serum pre-incubated to prevent non-specific binding.

The cultures were incubated for 3 to 9 days with primary antibody in 1% buffered BSA solution. After washing for four to five days in buffer, the cultures were incubated with a secondary antibody (goat anti-mouse IgG, CY3) for three to five days and then washed for four to five days. The explants were placed on slides, viewed for fluorescence and photographed. The controls passed Explants that were only incubated with the secondary antibody. Primary antisera (Boehringer, Mannheim) were monoclonal antibodies, which were diluted into working solutions of 0.5 μg / m and 1 μg / ml for anti-GAP-43 and synaptophysin.

Nissl stains were used to assess the morphology of the explants. The cocultures were fixed, dehydrated, stained in toluidine blue, placed on slides and photographed.

The explants cultured on microelectrode arrays were recorded at various times with regard to spontaneous electrical activity, at the earliest after 4 DIV (days in vitro) to at the latest 35 DIV in normal culture medium at a temperature of 35 ° C. Using a multichannel recording system, the 60 microelectrodes could be recorded simultaneously, which made it possible to test correlated activity in the cocultures. The restoration of functional links between the explants was shown. The electrodes had impedances of 100 to 300 kΩ (at 1 kHz), a distance of 500 μm and a diameter of 10 μm. The activity was recorded at 10 to 25 kHz per channel, stored and analyzed offline for latency.

While the explants with carbogen-equilibrated ACF, consisting of (each in ml / 1) 125 NaCl, 3.5 KC1, 1.3 MgS0 ₄ , 1.2 KH ₂ P0 ₄ , 2.4 CaCl ₂ , 26 NaHC0 ₃ , 10 glucose (pH 7.4), perfused at a flow rate of approximately 0.5 ml / min, the neuron sensitivity was tested for various channel blockers and receptor agonists and antagonists. The explants were discarded after the pharmacological experiments. 5 shows an electrical stimulation of electrogenic cells in the cell cluster on a microelectrode array, as was used in the experiments described here. 5a shows simple responses in an organotypic hippocampal culture after 17 days in vitro with monopolar electrical stimulation via the MEA electrode marked with an asterisk. Stimulation artifacts are not shown.

5b shows a long-term stimulation over more than one hour, in which uniform responses were obtained on the different electrodes. Each column shows the amplitude of the stimulus response to stimuli that was applied every 60 seconds to the electrode shown with a star.

Example 4: Testing FK506

FK506 - an immunosuppressor - that has been shown to have neuroprotective and neuroregenerative effects in the central and peripheral nervous system (Brecht and Herdegen, 1999, The New 'Twist': Inhibition of FKBP Rotamases as a Neuroregenerative and Neuroprotective Principle, Neuroforum 5: 36-43), was added to the culture medium at 0 DIV in a concentration of 50 nM and administered again with each exchange of the culture medium to 14 DIV. The cocultures were tested for correlated activity and the percentage of explants that showed correlation across an entire group of treated explants was compared to controversies where no explant was treated with the drug.

In the untreated control (n = 28), correlated activity was shown in approx. 60% after 14 days in vitro, during a correlated activity of almost 90% was demonstrated in the explants treated with FK506 (n = 18). In this context, “correlated activity” is understood to mean the regeneration of functional connections between the sections.

Claims

claims

1. Use of an electrode array to investigate regeneration in biological material promoted by active substances or physical processes.

2. Use according to claim 1, in which a microelectrode array is used as the electrode arrangement.

3. Use according to claim 1 or 2, in which the active substances are selected from the group: organic or chemical compounds and biologically active substances, such as peptides, proteins, nucleic acids.

4. Use according to one of claims 1 to 3, is examined in the regeneration of intercellular connections.

5. Use according to one of claims 1 to 4, in which the functional interconnections or re-interconnections between neutrons are examined.

6. Use according to claim 4 or 5, in which a time course of the resulting links or re-interconnections is examined.

7. Use according to one of claims 4 to 6, in which the regeneration of cells damaged with regard to their intercellular connection is examined.

8. Use according to one of claims 1 to 7, in which the regeneration of cultures and cocultures is observed repeatedly over a longer period of time, preferably more than one week.

9. Use according to any one of claims 1 to 8, wherein the biological material comprises juvenile cells.

10. Use according to any one of claims 1 to 8, wherein the biological material comprises cells in the adult-like state.

11. Use according to one of claims 1 to 10, in which reactions of receptor systems to administration of the active ingredients are examined.

12. Method for the investigation of pharmaceutical effects on the regeneration potential of functional connections in cultures, in which an electrode array is used.

13. The method according to claim 12, wherein an electrode array is used as in one of claims 1 to 11.