JPH0741081B2 - Nerve cell connection method - Google Patents

Description

TECHNICAL FIELD The present invention relates to a nerve cell binding method for binding nerve cells in a living body to each other.

[Conventional technology]

At present, little is known about the mechanism of regulation of nerve development. However, there are some reports on external or internal factors, and as an example of the external factor, the nerve growth factor (NGF) of sensory nerve contained in mouse tumor is known. There is.

This will be explained with reference to FIG. 4. When a target cell is present in the vicinity of a sympathetic ganglion neuron, NGF is secreted from the target cell (shown in FIG. 4 (a)). Then, the axon extends from the neuron (shown in FIG. 2 (b)), finally reaches the target cell, and both are bound (shown in FIG. 2 (c)).

By sprouting and extending the side branch from the axon of the nerve cell,
The mechanism of nerve synaptic connection is explained as shown in FIG. As shown in (a) of the figure, it is assumed that the nerve cells A _{1 to} D ₁ are connected to the muscle fibers A _{2 to} D ₂ . At this time, if the nerve cells C ₁ and D ₁ are cut for some reason, the muscle fibers C ₂ and D ₂ no longer respond to the stimulus.
However, after a lapse of time, the side branch buds from the axons of the nerve cells C ₁ and D ₁ , gradually expands, and joins to other nerve cells in about 2 weeks.

[Problems to be Solved by the Invention]

However, because of such a natural mechanism of neural cell connection, it takes a long time of 2 weeks, which is extremely inconvenient for experiments using biological tissues taken out of the living body, treatment of animals, and animal experiments. is there. Therefore, it is desired to realize smooth connection of nerve cells.

An object of the present invention is to provide a nerve cell binding method capable of overcoming the drawbacks of the prior art and enabling easy and reliable binding of nerve cells.

[Means for Solving the Problems]

The present inventor speculates that if Schwann cells of two nerve cells to be bound are fused, the binding can be suitably performed,
Further, in general, the present invention has been completed, focusing on the fact that cells having an outer shell can be fused by contacting and applying a high-voltage pulse.

That is, the nerve cell binding method according to the present invention is a Schwann cell of one nerve cell and a Schwann cell of the other nerve cell to be bound thereto in a biological tissue taken out of a living body or in a living body other than the human body. Are brought into contact with each other or sufficiently close to each other by a physical external force, and a first electrode is arranged on one nerve cell side and a second electrode is arranged on the other nerve cell side so as to sandwich these Schwann cells, and the first and It is characterized in that a pulse voltage that allows Schwann cells to fuse with each other is applied between the second electrodes. Here, the pulse voltage may be continuously applied multiple times.

[Action]

According to the present invention, two Schwann cells are brought into contact or sufficiently brought close to each other by a physical external force, and then a pulse voltage is applied. Here, the degree of application of the pulse voltage is
Since the electric field strength, the pulse width and the number of times of application are such that Schwann cells fuse, two nerve cells are instantly fused with each other. Therefore, they are bound to each other in Schwann cells within a short time after the fusion.

〔Example〕

 Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

FIG. 1 is an explanatory view of the method of the example, and schematically shows the mechanism of connecting nerve cells by a high-voltage pulse current. First, as shown in FIG. 4A, it is assumed that the nerve cell on the right side is cut between the second Schwann cell and the third Schwann cell for some reason. In this case,
Using a jig like tweezers, the nerve cells are bound by applying a physical external force. That is, the two Schwann cells of the two nerve cells are sandwiched by using the sandwiching members 21a, 22a and the sandwiching members 21b, 22b and brought into contact with each other on the side surfaces as shown in FIG. Let Then, while being held, a high voltage pulse voltage is applied once or several times between the first electrode 11 behind the holding members 21a, 22a and the second electrode 12 behind the holding members 21b, 22b. To do. Then, as shown in FIG. 7C, the outer shells of Schwann cells are broken and fused with each other. Such cell fusion can be generally realized by applying a high voltage pulse having an electric field strength of about several kV / cm and a time width of about several tens to several hundreds of microseconds about three times. Needless to say, cell fusion does not mean that axons are immediately connected and nerve cells are connected, and that it is necessary to leave the cells for a certain period of time after fusion.

FIG. 2 shows an example of an electrode set used for fusion of nerve cells by the method of the above-mentioned embodiment, FIG. 2 (a) is a diagram showing a partial cross-section of the entire structure, and FIG. 2 (b) to (f) are It is the principal part enlarged view. As shown in FIG. 3A, a first electrode 11 which is one of the electrodes is fixed to the tip of the main body case 10 at the base end, and a second electrode which is the other electrode is arranged in a position parallel to the first electrode 11.
The electrode 12 of is arranged. The base end of the second electrode 12 extends into the main body case 10 and is rotatable about a shaft 13 fixed to the main body case 10. Further, the lever 14 side opposite to the electrode side is biased clockwise by a spring 15 having one end fixed inside the main body case 10, and by a micrometer 16 screwed into a screw hole of the main body case 10,
The position is adjusted. Further, the first electrode 11 and the second electrode 12 are connected to the power source E, and the voltage application is controlled by the switch SW.

2 (c) and (e) show the first electrode 11 and the second electrode.
Twelve main parts are shown, and they have the same symmetrical structure. Part (c) of the same drawing is shown in section, and part (b) of the same drawing.
Is a sectional view taken along the line A ₁ -A ₂ , and FIG.
The electrode 12 is a view from A ₂ direction. Also, FIG.
FIG. 3B is a view of the second electrode 12 of FIG. 6E viewed from the A ₃ direction. As shown in the figure, the first electrode 11 and the second electrode 12 each have a needle-like member 41 such as platinum (Pt) in the center, and the needle-like member 41 is covered with an insulating cover member 42. There is. Then, at the same position on the facing surfaces of the first electrode 11 and the second electrode 12, the insulating cover member 42 is shaved off into a semi-cylindrical shape to form a recess 43, and the recess 43 has a circular shape at the center thereof. A through hole 44 having a shape is formed, and the needle-shaped member 41 is exposed here.

When the electrode having the above structure is used, cell fusion for connecting nerve cells can be very suitably performed. This will be described with reference to FIG. First, as shown in FIG. 4A, the main body case 10 is moved in the direction of arrow B in a state where the distance between the first electrode 11 and the second electrode 12 is increased. Then, after sandwiching the Schwann cell of the two nerve cells to be combined, the micrometer 16 attached to the main body case 10
Is turned to bring the first electrode 11 and the second electrode 12 close to each other. The above operation is continued until the positional relationship shown in FIG. That is, the two Schwann cells are bundled between the concave portions 43 of the first electrode 11 and the concave portions 43 of the second electrode 12, and the two Schwann cells are continuously contacted. Then, the switch SW is operated to apply the pulse voltage between the first electrode 11 and the second electrode 12. As a result, the Schwann cells are fused with each other, but the needle-shaped member 41 is not blocked by the insulating cover member 42 and does not touch the Schwann cells, so that the Schwann cells are not damaged.

Next, the present inventor conducted a binding experiment on nerve cells of a living mouse. That is, in the electrode set having the structure as shown in FIG. 2, an acicular member 41 made of platinum is prepared,
Two nerve cells were bundled into one. And 100 at 1 kV / cm
An electric pulse of μsec was applied three times. After 5 hours, when one nerve cell was electrically stimulated, a reaction appeared in the other nerve cell, so that binding could be confirmed.

[Effects of the Invention] As described above, in the present invention, two Schwann cells are brought into contact with each other or sufficiently brought close to each other by a physical external force, and then a pulse voltage is applied. Fused to each other. For this reason, since they are bound to each other in the Schwann cell within a short time after the fusion, easy and reliable nerve coupling can be realized.

[Brief description of drawings]

FIG. 1 is a diagram schematically showing fusion of nerve cells by a high-voltage pulse current according to an embodiment of the present invention, FIG. 2 is a diagram showing an electrode structure used in the embodiment, and FIG. 3 is a nerve cell of the embodiment. FIG. 4 is a diagram specifically showing fusion, FIG. 4 is a diagram showing a mechanism of nerve development, and FIG. 5 is a diagram showing changes in synaptic connections due to sprouting of axons. 10 ... Main body case, 11 ... First electrode, 12 ... Second electrode, 41 ... Needle-like member, 42 ... Insulating cover member, 43 ...
… Recesses, 44… Through holes.

Claims (2)

[Claims] 1. A Schwann cell of one nerve cell in a living body other than the human body and a Schwann cell of the other nerve cell to be bound thereto are brought into contact with each other or sufficiently close to each other by a physical external force. A first electrode may be disposed on the side of the one nerve cell and a second electrode may be disposed on the side of the other nerve cell so as to sandwich the Schwann cell, and the Schwann cells may be fused between the first and second electrodes. A method for coupling nerve cells, characterized in that a pulse voltage of a certain degree is applied. 2. The nerve cell coupling method according to claim 1, wherein the pulse voltage is continuously applied a plurality of times.