JPH01126532A - Polynuclear nmr probe for local measurement - Google Patents

Abstract

PURPOSE:To detect the NMR signal from the arbitrary local area of a live small animal, by providing a support stand, a fixing frame and a coil. CONSTITUTION:A small animal is mounted on the support stand 2 of a probe 1 in such a state that the back thereof is fixed to the animal receiving part 2a of said support stand 2. Thereafter, the trachea is cut open to insert a tracheal cannula in the trachea and artificial breathing is subsequently applied to the animal from a Teflon tube and the breast is incised to expose the heart. Next, a coil 5 is closely brought into contact with the left ventricle of the heart to be connected to the matching/tuning circuit 4 of a fixing frame 3 and the X-, Y- and Z-directions of the fixing frame 3 are regulated so as to hold the close contact state of the coil 5 to fix the fixing frame 3 to the support stand 2. After this setting, the support stand 2 is covered with a cover and the probe 1 is inserted in the vertical wide caliber superconductive magnet of a measuring system to be fixed thereto. By moving the coil 5 to an arbitrary local area, the NMR signal from the arbitrary local area of the live small animal can be detected.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a multinuclear NMR probe for local measurement;
Specifically, it concerns a probe suitable for detecting multi-nuclide NMR signals by placing a movable antenna in close contact with any local tissue of a living small animal, and improving the function and metabolism of local or whole organs through NMR measurements. This would make it possible to accurately evaluate drugs.

Conventional technology In the early days of this type of NMR measurement method for biological systems, a living animal was placed inside a large-diameter superconducting magnet that generated a high magnetic field, and a partial signal within the detection range was extracted. .

However, in this method, the detection signal includes skin, muscle, or all different organs, making it difficult to interpret the results from both a basic and applied point of view.

In order to solve the above drawbacks, an antenna using a coil is installed in a static magnetic field, a measurement part is located at the position of the antenna, and a local magnetic field is generated in the coil.
A device has been provided in which the NMR signal from a limited portion of the living body is obtained within the coil. However, in this device, it is technically difficult to match and tune the magnetic field within the large-diameter magnet and the weak magnetic field created by the coil, so the coil is fixed to the probe body that is inserted into the large-diameter magnet. Therefore, the measurement sites are extremely limited, such as the brain or muscles close to the body surface.

However, for example, in a pathological state, the lesions naturally exist in each region, and it is necessary to measure each region. Furthermore, detection of signals from target organs, i.e., any local area, is necessary for basic research in pathology, physiology, and biochemistry, and for evaluating drug efficacy, but as mentioned above, conventional devices cannot measure any local area. It was possible.

Purpose of the Invention The present invention has been made in view of the above-mentioned conventional problems.
The purpose of this invention is to enable detection of NMR signals from any location of a living small animal.

In order to achieve the above purpose, a movable coil (antenna) can be moved to any local location, but since the static magnetic field differs slightly within the magnet, it is difficult to move the coil to detect NMR signals. After moving, the magnetic field created by the coil must be aligned and tuned again, and the parts that perform this alignment and tuning must be inserted into the magnetic field, so it is required not to disturb the homogeneity of the magnetic field. be done.

Therefore, the present invention is intended to enable the detection of NMR signals such as 1H131p by moving a movable coil to an arbitrary location while solving the above-mentioned requirements.

Structure of the Invention In other words, the present invention provides a support base made of acrylic resin for storing a live small animal, and a support base made of acrylic resin that accommodates a small live animal, and a probe inserted and fixed into a superconducting large-diameter magnet. a fixed frame that can be freely adjusted in position in three dimensions; a capacitor and a matching box that constitute a matching/tuning circuit that are fixed to the fixed frame; and a support base that is detachably connected to the capacitor; A loop antenna consisting of a coil that is placed in close contact with any local tissue of a small animal housed in When the probe is inserted and fixed into a superconducting large-diameter magnet, the tuning knob can be operated from outside the large-diameter magnet, and the probe is configured to be operable from outside the large-diameter magnet. From the organization 1H131p,
l@p, 15 N, l 3 C,! It is an object of the present invention to provide a multi-nuclear NMR probe for local measurement suitable for detecting NMR signals of multi-nuclear species such as 3 Na.

K1 carp and below, multi-nuclide NMR signals from any local tissue of a living small animal (rat) according to the present invention, especially 1H1
``A multi-nuclear NMR probe used in an NMR apparatus suitable for detecting P-NMR signals will be explained in detail with reference to embodiments shown in the drawings.

Although not shown, a multinuclear NMR probe 1 using a movable antenna for local measurement according to the present invention shown in FIG.
Vertical normal diameter superconducting magnet NMR (manufactured by JEOL, JNM-G
It is used by inserting and fixing it into an NMR apparatus consisting of X200.4.8 Te5la, bore diameter 70 mm).

The probe 1 includes a cylindrical support base 2, a fixed frame 3 attached to the support base 2 so that its position can be adjusted in three dimensions, a matching/tuning circuit 4 attached to the front side of the fixed frame 3, and a matching/tuning circuit 4 attached to the front side of the fixed frame 3. A loop antenna 5 consisting of a surface coil is detachably connected to the tuning circuit 4, a base material 6 is attached directly above the antenna 5, and the probe is connected to the matching/tuning circuit 4! The main component is a tuning knob 7 that allows tuning operation from the outside while the device is placed in the NMR apparatus.

The support stand 2 is made of a non-magnetic transparent acrylic resin pipe (outer diameter 70+n+nφ, total length 540mm, wall thickness 5m).
m), cut off about 172 mm on one side of the cylindrical section along the axial direction by about 273 mm of the total length to accommodate a rat as a laboratory animal, and provide an animal storage section 2a in a semi-cylindrical shape, and on one end surface in the axial direction. It has a shape in which a fixing flange 10 (outer diameter 170 mmφ) is attached. A large number of recesses 2b for adjusting the position of the fixed frame are formed at predetermined intervals on the upper surface of the side wall of the animal storage section 2a of the support stand 2. Further, a urethane tube 11 for artificial respiration is attached to the inner wall of the support base 2.

The reason why the support base 2 is made of a non-magnetic material is that when it is inserted into a superconducting magnet, if it is made of a material that is even slightly magnetic, the magnetic field will be disturbed within the magnet. The above-mentioned acrylic resin is most preferably used because it cannot maintain uniformity, and because it is advantageous in terms of handling such as weight and ease of processing.

A fixed frame that can be attached to the support base 2 in a three-dimensional manner such as vertical direction (X direction in the figure), horizontal direction (Y direction in the figure), and height direction (X direction in the figure). As shown in Fig. 2, 3 is made of non-magnetic transparent vinyl chloride and is formed into a semicircular arc shape in cross section, and the - side end thereof is bent horizontally outward and brought into contact with the support base. A section 3a is provided.

The support base abutment portion 3a is provided with notches at predetermined intervals in the axial direction to form a large number of convex portions 3b, and the position of the convex portions 3b is freely adjustable in the X direction in the concave portion 2b of the support base 2. It is designed to fit. Furthermore, circumferential long holes 3c, 3c are bored in the other end of the fixed frame 3 on both sides in the axial direction, and the long holes 3
Screw in any position of c! A fixing plate 13 is attached via 2. A long hole 13a that is long in the axial direction is provided at the tip of the fixing plate 13, and screw holes 13 are provided at both ends of the long hole 13a.
It forms b. That screw hole! A screw 14 is inserted into 3b to press and fix it to the outer surface of the side wall of the support base 2. In this way, the fixed frame 3 is attached to the support base 2 via the fixed plate I3, and by adjusting the position of the fixed plate 13 in the circumferential direction with respect to the fixed frame 3, the fixed frame 3 is attached to the support base 2 in the Y direction. and position adjustment in the X direction.

The matching/tuning circuit 4 includes a non-magnetic trimmer capacitor 01 and a C
, and a matching box 14 constituting a matching circuit, and these trimmer capacitors CI, C2 and the matching box are fixed to the upper inner wall of the fixed frame 3. Matching box 14 of the matching/tuning circuit 4
is detachably connected to a spectrometer (not shown) during measurement. In addition, each of the above capacitors C1 and C2 has
Female connectors 15A and 15B are connected, respectively, and these connectors 15A and 15B are detachably connected to male connectors provided at the tips of conductive wires 17 extending from both ends of the antenna 5 (hereinafter referred to as surface coils). I try to measure it. By the combination of the coil 5 and the matching/tuning circuit 4, the 31p resonance frequency is 80.75, as will be described later.
It is set so that it can be tuned at an image frequency of 199.65M1lz.

The reason why the matching/tuning circuit 4 has the above configuration is that in a superconducting magnet (4.8 Tesla), the resonant frequency of 31p is 80.7.
On the other hand, in order to obtain a good P-NMR signal, the magnetic field inside the superconducting magnet must be in the best condition.For this reason, prior to the measurement of 31P-NMrt, a +H signal is used. This is because it is necessary to make SIIIM alignment, that is, to make the magnetic field in the best condition, and at that time, it is necessary to make the same coil resonate with the 'If column of 199.65 MIIz.In addition, using a trimmer capacitor as a capacitor The reason is that high frequency loss is extremely low,
This is also because the voltage resistance (approximately 5000V) is high.

Also, as will be described later, the matching circuit of the matching box 14 is better if it is shared for 3+p and 'H because the Q value decreases less and it is easier to operate if it is shared. It is. From the viewpoint of operability, a brass round rod 20 of 3 mm diameter is connected to the variable capacitor of the matching/tuning circuit 4 through a thick Teflon tube, and an operating shaft 22 is connected to the round rod 20 through a coupling 2I. The operating shaft 22 is connected to the fixed frame 3 and protrudes outward from the fixed frame 3, and a tuning knob 7 is provided at the tip thereof. Therefore, the tuning knob 7
This makes it possible to easily perform tuning operations from outside the NMR apparatus.

The surface coil 5 doubles as a high-frequency irradiation and detection coil for causing magnetic resonance, and non-magnetic materials with good electrical conductivity, such as Cu, Pt, and Ag, have good high-frequency characteristics. is used to form the coil.

The coil 5 is a heat-shrinkable Teflon tube 2 made of an insulator.
The reason for this is that when a coil without insulation coating is brought into close contact with an experimental animal (rat), high frequency loss (reduction in Q value) increases. Note that the thickness of the insulator is made thin because the thicker the insulator, the worse the S/N ratio becomes.

The number of turns and shape of the coil 5 are appropriately selected, and in this embodiment, as shown in FIGS. We are prototyping four types of coils: a 5CS coil with a single turn and a 5D coil with a single turn. A tuning circuit and a matching circuit when each of the above-mentioned coils 5A to 5D is used are as shown in the figure.

The resonance characteristics of the surface coil 5 are adjusted prior to measurement using a sweep generator I7, an oscilloscope 18, and a frequency meter 19, as shown in FIG.

An HMPA solution is used as the external reference substance 6 for the spectrum, which is placed directly above the surface coil 5 at a predetermined distance.As shown in FIG. , After sealing both ends with Araldite, acrylic resin holder 27
I'm putting it in.

The conductive wire I7 extends from both ends of the coil 5 through the holder 27.
It is fixed directly above the coil 5. In this way, the boulder 27 containing the base drop substance 6 is attached, and the male bottle connector 28 is attached to the tip of the conducting wire 17 with the coil 5 formed at the tip, which can be replaced with the female connector of the tuned circuit 4. is connected to. Since the conducting wire 17 provided with the coil 5 and the reference material 6 is itself flexible, the position of the coil 5 can be easily adjusted, and the fixed frame 3 to which the coil 5 is attached can be adjusted in three dimensions. , the coil 5 can be installed at any position and brought into close contact with the area to be measured.

Next, a case will be described in which in vivo "P-NMrt measurement of a rat heart is performed using the NMrt apparatus using the probe 1 described above.

Male rats (weight 250g to 350g) as experimental animals
The rat was anesthetized with bendoparbital (60 m
g/kg i, p) After that, the animal was placed in the animal housing part 2a of the support stand 2 of the probe 1 with the dorsal position fixed. After that, the trachea was incised and a tracheal cannula was inserted, followed by artificial respiration (97% 0.+3% Co, 65 times/min) through the Teflon tube 11.
), and under artificial respiration, the chest was opened and the heart exposed. After the coil 5 is brought into close contact with the left ventricle of the heart, it is connected to the matching/tuning circuit 4 of the fixed frame 3, and the fixed frame 3 is moved in the X, Y, and Z directions so as to maintain the close contact state of the coil 5. Adjust and
A fixed frame 3 was fixed to a support base 2. After setting in this manner, the support base 2 was covered with a copper cylindrical cover (not shown), and the probe 1 was inserted and fixed into the vertical wide-diameter superconducting magnet 40 of the measurement system shown in FIG. In Fig. 7, 4
! is the S 0M200 console, 42 is the gas cylinder, and 43 is the ventilator.

"P-NMR measurements were performed at a measurement frequency of 80.75 Mz, 14.0 μs during the pulse, and repeated integration at 1.9 sec intervals. ) are shown in Table 1 on the next page.

(Left below) Namely, (a) Enamel coated copper wire (0.5 m) shown in Figure 4 (I)
mφ) ring shape with 5 to 6 turns and diameter 8ffIIIlφ
When using the coil 5A and the matching/tuning circuit 4A shown in Fig. 4 (1) connected to the coil 5A, the Q of the coil decreased significantly, and no detection signal was obtained even after 1000 integrations (about 35 minutes). I couldn't.

(b) Using a coil 5B with a diameter of 14+nmφ wound with Teflon tube-coated copper wire (0.9mm+φ) 3 to 4 times as shown in FIG. 4(It), and a matching/tuning circuit 4B connected to the coil 5B, Although a decrease in the Q of the coil was observed, a signal was detected for the first time after 1000 integrations, although the S/N ratio was poor.

(C) When a coil 5C shown in FIG. 4 (III) is wound twice with a Teflon tube-coated silver wire (0.5n+mφ) to a diameter of 9mmφ, and a matching/tuning circuit 4C connected to the coil 5C is used. Good S/ after 200 integrations (about 7 minutes)
A signal could be detected with the N ratio.

(d) When using a surface coil 5D with a diameter of 9+++n+φ made of one turn of Teflon tube-coated silver wire (+mmJ) shown in FIG. 4(IV) and a matching/tuning circuit 4D, the Q of the coil is
There was almost no decrease in the value, and after 200 integrations (about 7 minutes),
An extremely good signal as shown in FIG. 8 was obtained.

In the figure, each signal is: l is β-ATP, 2 is α-ATp +
a −A D P + phosphodieste
rs, 3 is 7-ATP+β-ADP, 4 is Cr-P,
5 is Pi+2-phosphate of2.3-di
phosphoglycerate, 6 is 3-pho
sphate or2.3-diphosphogly
cerate, 7 is I (M P A. The above Cr
- If P is the standard (Oppm), the chemical shift is P
i; 5.3 ppm, β-ATP; -16.2 pl)
m, which corresponds to the existing chemical shifts of Pi and β-ATP (5.19 ppm, respectively).

-la, 13 ppm), and C
The half value of r-P and β-ATP is 0.8 pp each.
m and 1.4 ppm, which matched very well with the existing installation, and there was no problem in fixing the signal. In addition, the S/N ratio (
(or resolution) is also very good, and there is no problem in its quantitative nature. Furthermore, the ratio of Cr-P to ATP content, which is an index of the energy state of the myocardium, that is, ATP/Cr-P
The value was 0.67, which was in good agreement with the normal value in in vivo rat myocardium, and it was clear that the animal energy was good and there was no problem in specimen preparation.

As is clear from the above experimental results, the rat myocardium suggested that the surface coil 5 should be a one-turn coil 5D and a two-turn coil 5C, and a matching/tuning circuit/ID, 4G in combination with these. ``We were able to detect the P-NMrt signal with good sensitivity.

The above experimental results revealed the following regarding the coil 5, matching/tuning circuit 4, external reference material, etc.

Regarding the number of turns of the coil 5, the signal is detected more clearly when the number of turns is less than the number of turns.
Coil 5D) has the best S/
A signal was detected at the N ratio. This is because when the number of turns is large, the contact area with the myocardium becomes larger, that is, the electrostatic capacitance with the myocardium increases, and the Q value decreases further.

Regarding the wire diameter of the coil 5, it is related to the coil material, but strength, flexibility, ease of deformation, etc. are important.Myocardium is a pulsating organ, and in order to obtain a good S/N ratio, must be able to remain in close contact with the ventricular wall at all times during the beat. However, if the ventricle is pressed so strongly that it compresses the ventricle, the blood supply will be cut off to the compressed part of the ventricular wall, causing ischemia in that part. Although not shown in the data (table) above, according to the experimental results, a coil of pttmφ or AgO, 5n+mφ is easily deformed and a good S/N ratio cannot be obtained.
Furthermore, when the coil was wound 4 times with Cu having a diameter of 0.9 mm, the lower part of the coil was in ischemia.

In this experiment, the material and wire diameter of the coil 5 were Ag1, O
n+mφ was the most suitable.

Regarding the shape of the coil 5, if a single circular turn is used, if the inner diameter is too small, the signal amount will be small, and if the inner diameter is too large, it will protrude from the ventricular wall and no signal will be detected. 9-1 OfflIIIφ was the most suitable.

Regarding matching/tuning circuit 4, the matching circuit used for both 31p and 'H was superior, but this seems to be due to less reduction in the Q value of the coil, and it is easier to operate when the matching circuit is shared. It's easy too.

Regarding the reference substance, an internal standard substance is ideal, but since it is nearly impossible to introduce the standard substance into the heart in vivo, an external standard substance is used. HMPA as the standard substance is about + from the peak of Cr-P.
30 ppmil, and can be clearly identified without overlapping with the biological 31p signal.

The external reference substance must always be present at a fixed position with respect to the coil 5, and as a result of various experiments, the position directly above the coil 5 was found to be the highest position, as described above.

Effects of the Invention As is clear from the above explanation, the multi-nuclear NMR probe equipped with a movable antenna for local measurement according to the present invention has the following effects:
An antenna consisting of the above-mentioned support base, a fixed frame that is attached to the support base so that its position can be adjusted in a three-dimensional manner, a matching/tuning circuit attached to the fixed frame, and a surface coil that is replaceably connected to the matching/tuning circuit. The probe is equipped with an external reference material attached at a fixed position relative to the antenna, and a tuning knob for operation that is connected to the matching/tuning circuit and protrudes outward from the support base.
By installing it in the device, 'HN'P,'F,'N,+3G,' from any local tissue of a living small animal.
! NMR signals such as 3Na can be obtained clearly and easily. Specifically, it is possible to obtain the "P-NMrl signal" of an in vivo rat heart with high sensitivity, and therefore it is very useful for evaluating cardiac function and energy metabolism in physiology and pharmacology.

In particular, with the probe of the present invention, the movable antenna made of a coil can be moved three-dimensionally to fit the measurement area to any desired area to be measured, and can be brought into close contact with the measurement area. It is possible to detect various NMR signals from organs such as the brain, skeletal muscles, kidneys, liver, and even the beating heart, and has the advantage that measurement sites are not limited.

Further, when the probe is placed inside the superconducting magnet, fine adjustment of matching and tuning can be made using a protruding tuning knob, which has various advantages such as extremely simple operation.

[Brief explanation of the drawing]

Figures 1 (1) and (II) show examples of the probe according to the present invention, (r) is a schematic front view, (If) is a schematic plan view, and Figures 2 (■) and (■) are of the above probe. A fixed frame equipped with an antenna and a matching/tuning circuit attached to a support stand is shown, (I) is a schematic perspective view, (II) is a plan view, Figure 2 is a circuit diagram of the matching/tuning circuit, and Figure 4 ( D(11)(I[I
)(1%') is a schematic diagram showing the combination of four types of coils and the matching/tuning circuit set for each coil, Figure 5 is a tuning frequency adjustment circuit diagram, and Figure 6 is the installation state of the antenna and external reference material. 7 is a schematic block diagram of the NMR measurement system, and FIG. 8 is a diagram showing the results of P-NMR measurement of in vivo rat myocardium. !... Probe, 2... Support stand, 3...
Fixed frame, 4... Matching/tuning circuit, 5... Antenna (coil), 6... External reference material, 7... Tuning knob. Patent applicant: Takeda Pharmaceutical Co., Ltd. Representative Patent Attorney: Seihaku Hao and 2 others Figure 4 (I)

Claims (1)

[Claims] (1) A support stand made of non-magnetic material for storing a live small animal; a fixed frame that is attached to the support stand so that its position can be adjusted in three dimensions (vertical, horizontal, and height); and the fixed frame. A capacitor and a matching box constituting a matching/tuning circuit fixed to the capacitor and a coil detachably connected to the capacitor and brought into close contact with any local tissue of the small animal housed in the support base. a loop antenna, an external reference material attached to a fixed position with respect to the loop antenna, and a tuning knob connected to the matching/tuning circuit and protruding outward; A multinuclear NMR probe for local measurement suitable for detecting NMR signals of multiple nuclides from any local tissue of a small animal, the probe being inserted and fixed into a cell and configured such that the tuning knob can be operated in the inserted and fixed state.