JP7224642B2 - Measuring method and measuring device - Google Patents

Description

The present invention relates to a measuring method for measuring the state of nerve transmission and a measuring device for measuring the state of nerve transmission.

When a nerve cell receives an electrical signal, it transmits the signal to the end of the axon by changing the membrane potential and controlling the opening and closing of the sodium channel and the sodium channel. When the electrical signal reaches the end of the axon, a neurotransmitter is released that stimulates the next nerve cell to receive the electrical signal. Nerve transmission is performed by transmitting electrical signals in this way.

As an example of changes in membrane potential, there is research using nerve axons of spear squid (for example, Non-Patent Document 1). FIG. 8 is an example of changes in membrane potential, and FIG. 9 is a diagram showing potential propagation accompanying nerve activity, showing spatiotemporal changes in giant axons of a squid. It has been clarified that the membrane potential change associated with channel gating and the like is about 100 mV in a time frame of several milliseconds.

As a method of measuring membrane potential, a method of measuring potential by binding microelectrodes to cells is known (for example, Patent Document 1).

JP-A-09-024031

Raymond Chang, Physical Chemistry for Chemistry and Life Sciences, Tokyo Kagaku Doujin analytical chemistry, (US), 2014, vol. 86, No. 2, p. 1045-1052 Journal of Magnetic Resonance, (Ran), 2010, vol. 202, No. 2, p. 267-273

By measuring the state of nerve transmission, it can be applied to visualization of pain transmission and efficacy evaluation of analgesic action.
However, the existing potential measurement methods have application limitations such as invasiveness due to electrode insertion and measurement only at the probe installation site. In addition, the existing potential measuring method for measuring the site where the probe is installed has a problem that it is difficult to measure the potential in the deep part of the living body.
SUMMARY OF THE INVENTION It is an object of the present invention to provide a measuring method and a measuring device which are non-invasive, can measure even deep inside a living body, and can measure the state of nerve transmission.

As a result of earnest research to solve the above problems, the inventors of the present invention have found that the following inventions meet the above objects, and have completed the present invention.

一般式（１）において、Ｒ¹は、アルキル基、フェニル基、アミノ基、およびアザシクロアルキル基からなる群から選択されるいずれかを表し、Ｒ²、Ｒ³は、それぞれ独立に、アルキル基を表し、Ｒ⁴、Ｒ⁵は、それぞれ独立に、アルキル基、フェニル基、およびアルキルカルボキシル基からなる群から選択されるいずれかを表す。
＜５＞ 電子スピンを用いた磁気共鳴法により計測対象における神経伝達の状態を計測する計測装置であって、電位応答性分子を含む造影剤を含む前記計測対象に磁気共鳴法を適用して磁気共鳴信号を得る信号取得手段と、前記信号取得手段で得られた磁気共鳴信号から前記計測対象における神経伝達の状態を得る神経伝達状態取得手段とを含む、計測装置。 That is, the present invention relates to the following inventions.
<1> A measurement method for measuring the state of nerve transmission in a measurement target by a magnetic resonance method using electron spins, wherein the magnetic resonance method is applied to the measurement target containing a contrast agent containing a voltage-responsive molecule A measurement method, comprising: a first step of obtaining a resonance signal; and a second step of obtaining a neurotransmission state in the measurement object from the magnetic resonance signal obtained in the first step.
<2> The measurement method according to <1>, wherein the second step includes imaging the magnetic resonance signals obtained in the first step.
<3> The measurement method according to <1> or <2>, wherein the magnetic resonance method is electron spin resonance method or Overhauser MRI.
<4> The measuring method according to any one of <1> to <3>, wherein the voltage-responsive molecule is a molecule represented by the following general formula (1).

In general formula (1), R ¹ represents any one selected from the group consisting of an alkyl group, a phenyl group, an amino group, and an azacycloalkyl group; R ² and R ³ each independently represent an alkyl group; and R ⁴ and R ⁵ each independently represent one selected from the group consisting of an alkyl group, a phenyl group and an alkylcarboxyl group.
<5> A measuring device for measuring the state of neurotransmission in a measurement target by a magnetic resonance method using electron spins, wherein the magnetic resonance method is applied to the measurement target containing a contrast agent containing a voltage-responsive molecule A measuring apparatus comprising: a signal acquisition means for obtaining a resonance signal; and a nerve transmission state acquisition means for obtaining a state of nerve transmission in the measurement target from the magnetic resonance signal obtained by the signal acquisition means.

ADVANTAGE OF THE INVENTION According to this invention, the measuring method and measuring device which are non-invasive and can measure even the deep part of living bodies, and can measure the state of nerve transmission are provided.

The conceptual diagram of the measuring method of this invention is shown. FIG. 4 is a schematic diagram of integral EPR spectral shift and change in image intensity with potential change. FIG. 4 is a diagram showing a differential EPR spectrum with potential change; OMRI image at equilibrium potential. FIG. 4 is a diagram showing temporal changes in electron spin resonance spectrum intensity due to potential fluctuations of an electron-responsive molecule solution. It is a figure which shows the measuring device 1 which concerns on this invention. It is a figure which shows the measuring device 2 which concerns on this invention. It is a figure which shows the example of a membrane potential change. FIG. 4 is a diagram showing spatio-temporal changes in axons;

Embodiments of the present invention will be described in detail below. is not limited to the contents of

The measurement method of the present invention is a measurement method for measuring the state of neurotransmission in an object to be measured by magnetic resonance using electron spin, wherein the object to be measured contains a contrast agent containing a voltage-responsive molecule. The measurement method includes a first step of obtaining a magnetic resonance signal by application, and a second step of obtaining a neurotransmission state in the measurement target from the magnetic resonance signal obtained in the first step.

The features of the measuring method of the present invention are as follows.
(1) By magnetic resonance technique.
(2) using a contrast agent that detects potential changes;
(3) Detect potential changes by using spectral changes.
(4) It should be a technique for measuring or visualizing the intra-target propagation of potential change.

By using a contrast agent containing voltage-responsive molecules, changes in membrane potential caused by signal transmission in nerve transmission are followed, and the state of the voltage-responsive molecules changes. By applying the magnetic resonance method to a measurement target containing a contrast agent containing such voltage-responsive molecules, different magnetic resonance signals are detected depending on the state of the voltage-responsive molecules in response to changes in membrane potential. This magnetic resonance signal can be understood as a state of nerve transmission.

The measurement method of the present invention uses a magnetic resonance method and a contrast agent containing voltage-responsive molecules, and is a technique that can theoretically realize neurotransmission mapping of the entire body and deep parts of the body. Therefore, its application range is not limited to visualization of pain transmission and efficacy evaluation of analgesic action, but also relates to tactile sensation and random movement in general. The expected ripple effect is very large.

Preferably, in the measuring method of the present invention, the second step includes imaging the magnetic resonance signals obtained in the first step. By imaging the state of nerve transmission, it is possible to easily grasp the state of spatial nerve transmission of the measurement target.

The measurement method of the present invention can be used as a method for realizing real-time visualization of neurotransmission of terminal stimulation in a living body. The present invention creates a completely new measurement method that realizes real-time visualization of neurotransmission of terminal stimuli in living organisms by magnetic resonance using electron spins. Expected targets are visualization of pain transmission and efficacy evaluation of analgesic action. In addition, the measuring method and the measuring device of the invention can in principle measure the entire neural transmission activity in the deep part of the living body, and a wide range of ripple effects can be expected.

In the measuring method of the present invention, the magnetic resonance method is preferably electron spin resonance method or Overhauser MRI. By using these magnetic resonance methods, more accurate measurement images can be obtained.

Electron spin resonance (ESR or EPR) is more preferred. Preferably, the range of the magnetic field is 20 mT or less.

The magnetic resonance method is preferably a pulse method. The pulse method can be measured within a time of about several hundred microseconds to 1 millisecond, and can measure dynamic activity transition changes.

In the measuring method of the present invention, the voltage-responsive molecule is preferably a molecule represented by the following general formula (1). The molecule represented by the general formula (1) is preferable as the voltage-responsive molecule used in the measurement method of the present invention because it easily changes in milliseconds following the change in potential.

In general formula (1), R ¹ represents any one selected from the group consisting of an alkyl group, a phenyl group, an amino group, and an azacycloalkyl group; R ² and R ³ each independently represent an alkyl group; and R ⁴ and R ⁵ each independently represent one selected from the group consisting of an alkyl group, a phenyl group and an alkylcarboxyl group.

Further, the measuring device of the present invention is a measuring device for measuring the state of neurotransmission in a measurement target by a magnetic resonance method using electron spins, wherein the measurement target contains a contrast agent containing a voltage-responsive molecule. and a neural transmission state obtaining means for obtaining a state of neural transmission in the object to be measured from the magnetic resonance signals obtained by the signal obtaining means.
Moreover, the measuring device of the present invention can be used as a device that realizes real-time visualization of neurotransmission of terminal stimulation in a living body.
The measuring device of the present invention is suitable for carrying out the measuring method of the present invention.

[Measurement method of the present invention]
FIG. 1 shows a conceptual diagram of the measuring method of the present invention. The measurement method of the present invention uses the membrane potential response of a potential-responsive molecule "molecular electrode" probe in a living body, and uses a magnetic resonance method (for example, pulse ESR technology) that enables measurement and imaging on a time scale of about microseconds to milliseconds. and OMRI equipment) to realize measurement and imaging of neural activity and transmission processes. In addition, the measurement method of the present invention can be used to evaluate drug efficacy in disease models, develop molecular electrode derivatives, and the like. Imaging potential in milliseconds is extremely difficult to realize in terms of sensitivity, and can only be realized using the high-speed technology possessed by the inventor. . The measurement method of the present invention is developed for neural activity imaging by realizing high-speed dynamic potential measurement by speeding up the measurement system.

In the measuring method of the present invention, the state of nerve transmission is measured by acquiring changes in magnetic resonance signals according to changes in the membrane potential of the object to be measured, using voltage-responsive molecules and the magnetic resonance method.
Membrane potential is the potential difference that exists inside and outside the cell, and as described above, nerve cells transmit signals to axon terminals by changing the membrane potential and controlling the opening and closing of sodium channels and sodium channels. . As described above, in potential propagation accompanying nerve activity, potential fluctuations of about 120 mV occur in a time frame of about 5 ms. In order to measure and visualize this phenomenon deep inside the body, voltage-responsive molecules with a reaction speed of 1 s or less (preferably on the order of ms) and measurement/visualization techniques are required.

(measurement target)
The measurement target in the measurement target of the present invention is a sample or a living body (not limited to a mouse or the like). Specifically, living organisms such as humans, cows, horses, pigs, monkeys, guinea pigs, rabbits, rats, and mice can be targeted. Living bodies other than humans, such as cows, horses, pigs, monkeys, guinea pigs, rabbits, rats, and mice, may also be used. In addition, objects other than living organisms, such as cells and organs, can also be measured. The measurement method of the present invention is performed after administering or dripping a contrast agent containing electron-responsive molecules to these measurement objects. For example, when the measurement target is a living body, the administration method (administration route) of the contrast medium is not particularly limited, but the preferred contrast medium administration route is intravenous injection, skin application, intraperitoneal administration, or subcutaneous administration.

(contrast agent)
The contrast agent included in the measurement target of the present invention includes electron-responsive molecules. A voltage-responsive molecule is a molecule whose structure can be reversibly changed by a change in electric potential.
The electron-responsive molecule used in the present invention is used as a "molecular electrode" probe, has unpaired electrons, and changes in molecular structure following changes in the membrane potential of the measurement target, resulting in the generation of unpaired electrons. It is a molecule whose electronic state changes.
Specifically, the potential responsive molecule is preferably a molecule having a potential switching speed of 1 s or less. Note that the potential switching speed is the speed of molecular structural change caused by a potential change. The potential switching speed can be measured by a known method such as magnetic resonance method.

In order to measure the state of nerve transmission more accurately, the potential switching speed is preferably at the level of milliseconds (msec) or less, preferably 500 msec or less. The potential switching speed may be 300 ms or less, 200 ms or less, 100 ms or less, 80 ms or less, 50 ms or less, 30 ms or less, 10 ms or less, 5 ms or less, 1 ms or less, and the like. Also, the potential switching speed may be in the microsecond (μsec) level, and may be 50 μsec or more, 100 μsec or more, 500 μsec or more, and the like.

FIG. 2 is a schematic diagram of integral EPR spectral shift and image intensity change with potential change. For example, it is assumed that a sample A (for example, a molecule of structure A in formula (2) below) has a potential of 1 before binding of cations. Assume that the sample B (for example, the molecule of the structure B in the following formula (2)) has a potential of 2 after binding of the cations. The EPR spectrum of the sample with potential 1 (spectrum 1 of sample 1 in FIG. 2) changes to the EPR spectrum of the sample with potential 2 (spectrum 2 of sample 2 in FIG. 2) as the potential changes. At this time, if the measurement and imaging are performed under the conditions of the maximum absorption position of the spectrum (Condition 1 or Condition 2 in FIG. 2), the intensity change shown in the table of FIG. 2 is shown, so the potential can be imaged. . Voltage-dependent changes in electron spin resonance (ESR) spectral position can be obtained by using contrast agents that contain voltage responsiveness.

In the measuring method of the present invention, the structure of the voltage-responsive molecule is not particularly limited. A molecule represented by the following general formula (1) is preferable as the voltage-responsive molecule used in the measurement method of the present invention. A molecule represented by the following general formula (1) undergoes a structural change due to a change in electric potential, and can exhibit different magnetic resonance behavior. In addition, it is easy to change in millisecond level following changes in potential, and is therefore suitable for detecting changes in membrane potential.

In general formula (1), R ¹ represents any one selected from the group consisting of an alkyl group, a phenyl group, an amino group, and an azacycloalkyl group; R ² and R ³ each independently represent an alkyl group; and R ⁴ and R ⁵ each independently represent one selected from the group consisting of an alkyl group, a phenyl group and an alkylcarboxyl group.

Alkyl groups may be straight chain, branched or cyclic. Examples of alkyl groups include alkyl groups having 1 to 8 carbon atoms and 1 to 5 carbon atoms. Specific examples include a methyl group and an ethyl group.

The phenyl group may be unsubstituted or substituted.

An amino group is a functional group represented by "-NR ⁶ R ⁷ ", and R ⁶ and R ⁷ each independently include an alkyl group having 1 to 8 carbon atoms or 1 to 5 carbon atoms. Specific examples include an amino group, a dimethylamino group, a diethylamino group, a methylethylamino group and the like.

Azacycloalkyl groups are functional groups in which one carbon atom of a cycloalkyl group has been replaced with a nitrogen atom. Azacycloalkyl groups can be 4- to 10- or 5- to 8-membered rings. The azacycloalkyl group is preferably attached to a carbon atom of the imidazoline skeleton via the nitrogen atom. Specific examples include pyrrolidyl group, piperidyl group, azepanyl group, azocanyl group and the like.

An alkylcarboxyl group is a functional group in which an alkyl group represented by "--R ⁸ --COOH" and a carboxyl group are bonded. Examples of R ⁸ include alkyl groups having 1 to 8 carbon atoms and 1 to 5 carbon atoms.

In general formula (1), R ¹ is preferably any one selected from the group consisting of an alkyl group, a phenyl group and an azacycloalkyl group, more preferably an azacycloalkyl group.
In general formula (1), R ⁴ and R ⁵ are each independently preferably an alkyl group or a phenyl group, more preferably an alkyl group.

The molecule represented by the general formula (1) is a molecule in which a cation (X ⁺ ) can be chemically or coordinately bonded to the nitrogen atom at the 3-position of the imidazoline ring as shown in the following formula (2). is. Desorption of the cation to the nitrogen atom at the 3-position of the imidazoline ring changes the electron density of the nitrogen atom at the 1-position to which the oxygen radical binds (dotted line in formula (2)), resulting in Three electron spin resonance absorption positions change. This phenomenon can be used to measure potential.

Molecules represented by the general formula (1) include (4-amino-2,2,5,5-tetraethyl-2-imidazolin-1-yloxyl) radical, (4-(pyrrolidin-1-yl)-2 , 2,5,5-tetraethyl-2-imidazolin-1-yloxyl) radical, (4-(azepan-1-yl)-2,2,5,5-tetraethyl-2-imidazolin-1-yloxyl) radical and , Non-Patent Documents 2 and 3 can be used.

3 and 4 are experimental results leading to the present invention.

FIG. 3 is a stack of differential EPR spectra obtained by measuring two samples with different potentials in their static states, and is a differential EPR spectrum accompanying potential change. Measurement was performed under the following conditions.

Sample preparation:
4-amino-2,2,5,5-tetramethyl-3-imidazoline-1-yloxyl radical was used as the voltage-responsive molecule represented by general formula (1). 4-amino-2,2,5,5-tetramethyl-3-imidazoline-1-yloxyl radical was dissolved in phosphate buffer to a final concentration of 100 μM, and diluted with hydrochloric acid solution and sodium hydroxide solution to pH 6 or pH 8.0. 0. As a result, as samples with different potentials, 4-amino-2,2,5,5-tetramethyl-3-imidazoline-1-yloxyl radical (corresponding to structure A in the above formula (2)) and 4-amino- A sample of 2,2,5,5-tetramethyl-imidazolidine-1-yloxyl radical (corresponding to structure B in formula (2) above) was obtained.
・X-band measurement conditions:
JEOL X- band ESR JES-RE1X, magnetic field strength 3,330 Gauss, sweep width +/- 5 mT, sweep time 10 seconds, magnetic field modulation 100 kHz, 0.05 mT, microwave 9.45 GHz, 1 mW
・Magnetic field position in the spectrum:
The frequency is constant because the spectrum is acquired in magnetic field sweep mode.

As shown in FIG. 3, the X-band EPR spectrum changes from spectrum A to spectrum B as the potential changes. The magnetic field position on the right side of spectrum A is 3,345 GHz and the magnetic field position on the right side of spectrum B is 3,345.7 GHz.

FIG. 4 is a diagram showing experimental results of spectral imaging of a solution of voltage-responsive molecules. By performing spectral imaging of a solution of voltage-responsive molecules having different potentials, the potential was imaged for a (static) phenomenon of several seconds. In the OMRI image at the equilibrium potential shown in FIG. 4, the potential difference of each solution is 0, 90, 250 mV counterclockwise from the top.

FIG. 5 shows temporal changes in electron spin resonance spectrum intensity due to potential fluctuations of the electron-responsive molecule solution. Measurement was performed under the following conditions.

・Sample preparation:
4-amino-2,2,5,5-tetramethyl-2-imidazoline-1-yloxyl radical was used as the voltage-responsive molecule represented by general formula (1). 4-amino-2,2,5,5-tetramethyl-2-imidazoline-1-yloxyl radical was dissolved in phosphate buffer to a final concentration of 100 μM, and adjusted to pH 6 with hydrochloric acid solution and sodium hydroxide solution. .
・X-band measurement conditions:
JEOL X- band ESR JES-RE1X, magnetic field strength 3,345 Gauss fixed, magnetic field modulation 100 kHz, 0.05 mT, microwave 9.45 GHz, 1 mW
・Experimental operation:
A sinusoidal voltage having a period of about 60 Hz and a peak/peak voltage of 1 V was input from an external oscillation source (NF WF1973) to the electrodes connected to the X-band solution cell to modulate the solution potential. ESR output data were acquired at a sampling rate of about 50 kHz in time with the modulation.
ΔpH1 corresponds to about 50 mV. In a solution of pH 6, when 1 V was applied from the maximum spectral absorption position, which is the initial position, the potential response reached a plateau with this molecule, and the data change was measured by modulating it sufficiently beyond about pH 9 where it did not change.

As shown in FIG. 5, when the solution potential is changed, a corresponding decrease (move from maximum resonant absorption to non-resonant absorption) or increase (move from non-resonant absorption to maximum resonant absorption) in signal intensity is observed. This indicates that the voltage-responsive molecule responds to potential fluctuations on the order of milliseconds and gives information on the potential of the solution. It has not been reported whether this voltage-responsive molecule responds to fast fluctuations in electric potential or whether it can be visualized. The present inventors have found for the first time that the electrophoretic molecule has high responsiveness, and that the spectrum changes following the potential change at the millisecond level.

By measuring the ESR at the m-second level using the voltage-responsive molecule and continuously measuring the EPR, it is possible to obtain the electric potential information of the contrast agent in response to the electric potential fluctuation at the m-second level. .

[Measuring device of the present invention]
A measurement apparatus according to the present invention includes a magnetic resonance measurement apparatus, a control processing section, and a display section. The magnetic resonance measuring device is signal acquisition means, and the control processing unit is nerve transmission information acquisition means. Nerve transmission information can be obtained by analyzing the magnetic resonance signals obtained by the magnetic resonance measuring device by the control processing unit.

The magnetic resonance instrument may be an ESR instrument or an Overhauser MRI instrument. ESR devices may be either CW or pulsed. A pulse type is preferable because the measurement time can be shortened. The OMRI apparatus includes, for example, the apparatus disclosed in International Publication WO2010/110384. In other words, "magnetic field generating means for generating a magnetic field for exciting the magnetic resonance of the object to be measured, and moving means for moving the object to be measured or the magnetic field generating means to move the object to be measured in the magnetic field of the magnetic field generating means. Then, a gradient magnetic field is applied to either or both of the moving direction y of the object to be measured with respect to the magnetic field generating means and the direction x perpendicular to the moving direction y without stopping during movement by the moving means to phase-encode the object. and frequency encoding, a measurement means for obtaining a measurement image signal in the measurement object; "equipment having" can be used.

The control processing unit obtains the state of nerve transmission by performing processing such as Fourier transform on data based on magnetic resonance signals acquired by the magnetic resonance measurement device, or by performing imaging processing. Acquired data can be stored in the control processing unit, and data necessary for analysis can be read out.
Also, the control processing unit stores a control program and the like for controlling the measurement conditions and the like of the magnetic resonance measurement apparatus. The control processor can output a control program to control the measurement conditions and the like of the magnetic resonance measurement apparatus.

The display unit can display an ESR spectrum, an NMR spectrum, an analysis image, and the like obtained by processing in the control processing unit.

[Measurement method 1]
A measurement method 1 according to the present invention is an example of a measurement method using a measurement device 1 (see FIG. 6) that includes a pulse-type ESR device 100, a control processing unit 200, and a display unit 300. FIG.

A pulse-type ESR device 100 includes an external magnetic field generator 10 having a permanent magnet 10a, a magnetic field sweep coil 10b, and a magnetic field gradient coil 10c, an RF coil (resonator) 20, a pulse generator 30, and an RF pulse generator. 40 and a detection unit 50 .

The magnetic field sweeping coil 10b and the detection unit 50 are each connected to a control processing unit and can be controlled by commands from the control processing unit 200. FIG. The pulse generator 30 is also connected to the control processor 200 , the magnetic field gradient coil 10 c and the RF pulse generator 40 . The magnetic field gradient coil 10c and the RF pulse generator 40 are controlled by the pulse sequence generated by the pulse generator 30 according to the command from the control processor 200. FIG. The display unit 300 is connected to the control processing unit 200 and can display analysis data processed by the control processing unit 200 .

- 1st step The magnetic field sweep coil 10b is adjusted to set a predetermined static magnetic field in the range of 0 to 50 mT (for example, 20 mT). The pulse sequence used for measurement is set in consideration of the measurement site, S/N ratio, and the like. The measurement time is set to, for example, 1 s or less (100 μsec to 1 msec, 1 msec to 10 msec, 10 to 100 msec, etc.).
First, a mouse to be measured is placed inside the RF coil 20 . A contrast agent containing the voltage-responsive molecule A is administered to this measurement target mouse. Note that only a part of the mouse such as the head and tail can be used as the measurement site.
Next, the mouse in the RF coil 20 is irradiated with RF pulses (frequency of about 500 to 600 MHz) generated by a predetermined pulse sequence. Further, the magnetic field gradient coil 10c is driven according to a predetermined pulse sequence to generate gradient magnetic fields of a predetermined magnitude at predetermined times. An ESR signal due to electron spin resonance is detected by the detector 50 .
Measurements are continuously taken at predetermined intervals to acquire ESR signals over time. The measurement interval is set to, for example, 1 s or less (10 to 100 ms, 10 to 500 ms, etc.).

・Second step In the control processing unit 200, the ESR signal detected in the first step is subjected to processing such as Fourier transform, and data of the magnetic field range including the maximum spectral resonance absorption position of the potential responsive molecule A is extracted, To analyze. For the magnetic field position of the maximum spectral resonance absorption position of the voltage-responsive molecule A, for example, the ESR spectrum of the electron-responsive molecule A is measured in advance and data stored in the control processing unit 200 can be used.
Due to the potential change in the membrane potential, the voltage-responsive molecule A changes its structure, and the absorption intensity (signal intensity) at the maximum spectral resonance absorption position of the voltage-responsive molecule A decreases. By analyzing the absorption intensity of the voltage-responsive molecule A over time, it is possible to identify a portion where the absorption intensity of the voltage-responsive molecule A is low, which leads to visualization of pain. By analyzing the movement direction of the portion of the potential-responsive molecule A having low absorption intensity, the movement direction of the portion of the voltage-responsive molecule A having low absorption intensity can be determined as the nerve running direction. In addition, it can be determined that nerve transmission is occurring at a site where a decrease in the absorption intensity of the voltage-responsive molecule A is observed.
Alternatively, the ESR signal may be processed and imaged. It is also possible to visualize the state of nerve transmission from changes in the image over time (changes in portions with low signal intensity).

In measurement method 1, the first step can also be performed while changing the measurement site in the measurement target (acquiring ESR signal data while changing the location of the RF coil 20). As a result, useful information about neurotransmission can be obtained.

In the measurement method 1, the first step can also be simultaneous measurement at two or more points along nerve runs. Simultaneous measurement at two or more points in this way results in useful information on neurotransmission due to its data synchrony (the same information is transmitted after a certain delay).

In measurement method 1, position information can also be acquired. On the other hand, depending on the measurement site, a shorter measurement time may be required. If shortening of the measurement time is prioritized over acquisition of positional information depending on the measurement site and the purpose of measurement, measurement can be performed with the magnetic field gradient coil turned off.

Further, when shortening the measurement time is prioritized over acquisition of position information, the measurement method of the present invention may be performed using a measurement device having a pulse-type ESR device that does not have a magnetic field gradient coil. good. For example, an ESR device comprising an external magnetic field generator having a permanent magnet and a magnetic field sweeping coil, an RF coil (resonator), a pulse generator, an RF pulse generator, a detector, a control processor, and a display Measurement can be performed using a measuring device having a portion. The measurement method is the same as measurement method 1, except that the magnetic field gradient coil is not operated.

Even when the magnetic field gradient coil is turned off or the measurement is performed with an apparatus without a magnetic field gradient coil, measurement is performed while changing the measurement site of the measurement target (for example, several sites from the head to the tail of the mouse ), it is possible to determine the site where the change in the absorption intensity of the potential-responsive molecule A is observed and the site where it is not observed.
In addition, if two or more points are measured simultaneously, neurotransmission can be evaluated from the data synchrony (the same information is transmitted after a certain delay).

[Measurement method 2]
A measurement method 2 according to the present invention is an example of a measurement method using a measurement device 2 (see FIG. 7) including an OMRI (Overhauser MRI) device 101, a control processing unit 200, and a display unit 300. FIG.

The OMRI apparatus 101 includes a first external magnetic field generator 11 having a permanent magnet 11a, a magnetic field sweeping coil 11b, and a magnetic field gradient coil 11c, and a second external magnetic field generator 11 having a permanent magnet 12a, a magnetic field sweeping coil 12b, and a magnetic field gradient coil 12c. It comprises an external magnetic field generator 12 , an RF coil (resonator) 21 , a pulse generator 31 , an RF pulse generator 41 , a detector 51 , and moving means 61 .

The magnetic field sweeping coil 11b, the magnetic field sweeping coil 12b, and the detection section 51 are each connected to the control processing section 200 and can be controlled by commands from the control processing section 200. FIG.
The pulse generator 31 is connected to the control processor 200 , the magnetic field gradient coil 11 c, the magnetic field gradient coil 12 c and the RF pulse generator 41 . The magnetic field gradient coil 11c, the magnetic field gradient coil 12c, and the RF pulse generator 41 are controlled by the pulse sequence generated by the pulse generator 31 according to the command from the control processing unit 200. FIG.
The moving means 61 is means for moving the RF coil 21 or the first and second external magnetic field generators so that the RF coil 21 can move in the magnetic field of the first and second external magnetic field generators in order. is.
The display unit 300 is connected to the control processing unit 200 and can display analysis data processed by the control processing unit 200 .

- 1st step The magnetic field strength of the 1st external magnetic field generator 11 is set to a predetermined static magnetic field in the range of more than 0 and 50 mT or less (for example, 20 mT). The magnetic field strength of the second external magnetic field generator 12 is set to a value (for example, 1.5 T) greater than the magnetic field strength of the first external magnetic field generator 11 within the range of 0 to 11 T. Also, the pulse sequence used for measurement is set in consideration of the measurement site, the S/N ratio, and the like. For example, the measurement time of nuclear magnetic resonance (NMR) is set to 1 s or less (100 μsec to 1 msec, 1 msec to 10 msec, 10 to 100 msec, etc.).

First, the RF coil 21 with the mouse inside is placed in the magnetic field of the first external magnetic field generator 11 . A contrast agent containing the voltage-responsive molecule A is administered to this measurement target mouse.
Next, the mouse in the RF coil 21 is irradiated with an RF pulse (frequency of about 500 to 600 MHz) generated by a predetermined pulse sequence to excite electron spins.
After exciting the electron spins, the moving means 62 moves the RF coil 21 into the magnetic field of the second external magnetic field generator 12 . At this time, the movement should be several seconds or less (for example, 1 to 10 seconds).

After moving the RF coil 21 into the magnetic field of the second external magnetic field generator 12, RF pulses (frequency 700 to 800 MHz) are applied from the RF coil 21 to generate nuclear magnetic resonance. Also, by controlling the magnetic field gradient coil 12c according to a predetermined pulse sequence, a gradient magnetic field of a predetermined magnitude is generated at a predetermined number of times. NMR signals emitted by nuclear magnetic resonance are detected by the detector 51 .
The RF coil 21 is moved into the first external magnetic field generator 11 by the moving means 62, and similar operations are repeated to acquire NMR signals over time.

・Second step In the control processing unit 200, the NMR signal detected in the first step is subjected to processing such as Fourier transform, and data of the magnetic field range including the maximum spectral resonance absorption position of the potential responsive molecule A is extracted. ,To analyze.
As in the measurement method 1, by analyzing changes in the absorption intensity of the voltage-responsive molecule A, information on nerve transmission can be obtained. Alternatively, the NMR signal may be processed and imaged. It is also possible to visualize the state of nerve transmission from changes in the image over time.

As with the measurement method 1, the measurement method 2 may perform measurement while changing the measurement site in the first step, or may perform measurement at two or more points simultaneously.

In addition, the measuring method of the present invention is not limited to the measuring method 1 and the measuring method 2.
For example, in the first step of the measurement method of the present invention, the measurement apparatus shown in FIG. 9 is used to excite electron spins in the magnetic field of the first external magnetic field generator, and then A step of applying an RF pulse (frequency of 700 to 800 MHz) in the magnetic field of the first external magnetic field generator to generate nuclear magnetic resonance may be employed.

Alternatively, a measuring device having a CW ESR device instead of a pulse ESR device may be used to measure a measurement target containing a contrast medium containing voltage-responsive molecules. When performing measurement using a measurement apparatus having a CW ESR device, the first step can be a step of performing measurement by sweeping the magnetic field over a predetermined range while irradiating constant microwaves.

In addition, the first step of the measurement method of the present invention is to use a measurement device having a CW ESR device to measure 2 to 5 points including the magnetic field position of the maximum spectral resonance absorption position of the voltage-responsive molecule included in the measurement target. A magnetic field may be fixed at , and microwave irradiation and ESR signal acquisition may be performed at predetermined measurement intervals (for example, 1 second or less or 10 to 100 ms).

Moreover, the measuring device of the present invention is not limited to the measuring device 1 and the measuring device 2 . The measuring device of the present invention can be appropriately selected according to the object to be measured, the measuring method of the present invention, and the like. For example, a device without magnetic gradient coils may be used. Alternatively, a device whose magnetic field source is an electromagnet may be used. For example, an electromagnet may be used instead of the permanent magnet 10a of the ESR device 100, or an electromagnet may be used instead of the permanent magnets 11a and 12a of the OMRI device 101. FIG.

INDUSTRIAL APPLICABILITY The measuring method and the measuring device of the present invention can in principle measure all neural transmission activities in the deep part of the living body, and can be expected to have a wide range of ripple effects.

1, 2 measuring device according to the present invention 10 external magnetic field generator 11 first external magnetic field generator 12 second external magnetic field generator 10a, 11a, 12a permanent magnet 10b, 11b, 12b magnetic field sweep coil 10c, 11c, 12c Magnetic field gradient coils 20, 21 RF coils 30, 31 Pulse generators 40, 41 RF pulse generators 50, 51 Detector 61 Moving means 100 ESR device 101 OMRI device 200 Control processing unit 300 Display unit

Claims (5)

A measurement method for measuring the state of neurotransmission in a measurement target by a magnetic resonance method using electron spin,
a first step of obtaining a magnetic resonance signal by applying a magnetic resonance method to the measurement object containing a contrast agent containing voltage-responsive molecules;
and a second step of obtaining a neurotransmission state in the measurement object from the magnetic resonance signals obtained in the first step. 2. The measuring method according to claim 1, wherein said second step includes imaging the magnetic resonance signals obtained in said first step. 3. The measuring method according to claim 1, wherein the magnetic resonance method is electron spin resonance method or Overhauser MRI. The measuring method according to any one of claims 1 to 3, wherein the voltage-responsive molecule is a molecule represented by the following general formula (1).

In general formula (1), R ¹ represents any selected from the group consisting of an alkyl group, a phenyl group, an amino group, and an azacycloalkyl group;
R ² and R ³ each independently represent an alkyl group,
R ⁴ and R ⁵ each independently represent one selected from the group consisting of an alkyl group, a phenyl group and an alkylcarboxyl group. A measuring device for measuring the state of neurotransmission in a measurement target by a magnetic resonance method using electron spin,
a signal acquisition means for obtaining a magnetic resonance signal by applying a magnetic resonance method to the object to be measured containing a contrast agent containing a voltage-responsive molecule;
a nerve transmission state obtaining means for obtaining a state of nerve transmission in the measurement object from the magnetic resonance signal obtained by the signal obtaining means.

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