JPH0327225A - Novel test animal and production thereof - Google Patents

Abstract

PURPOSE:To obtain test animal useful for judging effect of remedy drug for Alzheimer type dementia by administering combined body of nerve growing factor and nerve cell toxin to brain cortex and/or hippocampus of mammal other than human and selectively destroying cholinergic neuron. CONSTITUTION:1.5-3 molecules of nerve growing factor (e.g. 2.5S.NGF) are reacted with a molecule of nerve cell toxin (e.g. diphtheria toxin) in the presence of water-soluble condensation agent [e.g. 1-ethyl-3-(3-L-dimethylaminopropyl) carbodiimide] in a solvent (e.g. water) at pH5.5-8.0 to obtain combined body of the nerve growing factor and nerve cell toxin. Next, 0.5-30mug said combined body is administered to brain cortex and/or hippocampus of mammal (e.g. rat) other than human and the mammal is bred for 3-7 day to selectively destroy cholinergic neuron projected to brain cortex from Meynert nucleus or bottom part of fore-brain and/or cholinergic neuron projected from septal body.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to a novel experimental animal useful as a dementia animal model and a method for producing the same. [Prior art] The average life expectancy of the Japanese people is among the highest in the world, and the proportion of elderly people in the total population is increasing year by year.
The number of patients with dementia of the lzheia + er ) type is also increasing. Pathologically speaking, this Alzheimer's type dementia is characterized by Meyner's dementia at the base of the brain.
) cholinergic neurons (Cholin) in the nucleus
It is characterized by a significant loss of ergic neuron. In order to develop drugs to treat Alzheimer's type dementia, pathological animal models are needed to determine drug efficacy. It is known that the basal forebrain has been destroyed by administering poisonous substances such as kainic acid, ibotenic acid, and ethylcholine mustard aziridinium ion (AF64A). However, both electrically disrupted animals and animal models treated with kainic acid or ibotenic acid do not contain non-cholinergic neurons or passing fibers.
) and other nervous system neurons other than cholinergic neurons are also destroyed, and in animal models administered with AF64A, cholinergic neurons other than cholinergic neurons that project from Meynert's nucleus to the cerebral cortex are also damaged indiscriminately. Therefore, none of these known animal models are necessarily suitable as animal models for determining drug efficacy. [Structure and Effects of the Invention] The present inventors have conducted extensive research in order to create experimental animals with dementia, especially animal models of Alzheimer's disease. )
When a conjugate of cholinergic neurons and a neurotoxin is administered to the cerebral cortex of an animal, the conjugate is taken up from the axon terminals of cholinergic neurons in the cerebral cortex, and is transferred to Meynert's nucleus (in primates) or the forebrain. Selective destruction of cholinergic neurons projecting from the basal region (in animals other than primates) to the cerebral cortex, and the extent of this destruction depends on the dose of the NGF-neuronotoxin conjugate. It was found that there is a dose-dependent relationship between the two, and that the degree of destruction can be controlled by adjusting the dose. Furthermore, the present inventors have also found that when the above conjugate is administered to the hippocampus of animals, cholinergic neurons projecting from the septal nucleus to the hippocampus are selectively and dose-dependently destroyed. .

Based on such findings, the present invention provides a method for producing non-human mammals in which cholinergic neurons projecting from Meynert's nucleus or the basal forebrain to the cerebral cortex and/or cholinergic neurons projecting from the septal nucleus to the hippocampus have been selectively destroyed. An animal and its production method.

Mammals to which the present invention is applied include, for example, primates such as monkeys, rodents such as rats, mice, and guinea pigs, dogs, and cats, and especially preferred are rodents such as rats and mice.

The animal of the present invention is produced by administering a conjugate of NGF and a neuronal cytotoxin to the cerebral cortex and/or hippocampus of the above mammal to induce cholinergic neurons that project from Meynert's nucleus or the basal forebrain to the cerebral cortex. It can be created by selectively destroying cholinergic neurons that project from the septal nucleus to the hippocampus. A conjugate of NGF and a neuronal cytotoxin can be prepared by reacting NGF and a neuronal cytotoxin. NGF includes human fetal vINGF, mouse submandibular It,
9NCF, snake venom NGF, guinea pig prostate NGF1 rabbit prostate NGF, bovine prostate NGF, bovine semen NGF
, musk rat submandibular gland NGF can be used, especially mouse submandibular gland NGF 7S・NGF, β-NGF.
, 2.5 3-NGF, etc. can be suitably used.

Further, as the nerve cytotoxin, for example, protein 1 toxin such as diphtheria toxin, cholera toxin, and ricin can be suitably used. These fluorescent white matter toxins are
Usually, it consists of A chain and B chain subunits, but in the present invention, any one containing Aim can be used.

The above reaction can be easily carried out in a suitable solvent in the presence of a condensing agent. During the reaction, it is preferable to use an excess amount of NGF relative to the neuronal cytotoxin so that no unreacted neuronal cytotoxin remains after the reaction is complete; for example, 1.5 to 3 NGF per molecule of neuronal cytotoxicity. Preferably, molecules are used.

As the condensing agent, condensing agents used in begutide synthesis can be used, such as 1-ethyl-3(3-L-dimethylaminopropyl)carbodiimide, N-cyclohexyl-N'-morpholinocarbodiimide, Water-soluble condensing agents such as these can be suitably used. Furthermore, water is preferred as the reaction solvent. The reaction proceeds suitably at room temperature, but
During the reaction, use an appropriate buffer such as phosphate buffer,
It is preferable to maintain the pH of the reaction solution at about 5.5 to 8.0.

After completion of the reaction, it is desirable to purify the resulting conjugate using methods commonly used in this field, such as dialysis and gel filtration.

The thus obtained conjugate of NGF and neuronal cytotoxin can be administered to the cerebral cortex or hippocampus by a conventional method,
For example, a hole may be made in the cephalic bone and the drug may be administered through this hole. More specifically, when administering to the cerebral cortex from the perforated part of the cephalic bone, several perforations are made in each cerebral hemisphere, centering around the cruciate suture of the two cranial bones, and the conjugate is administered from the perforated part to the cerebral cortex. This can be suitably carried out by injecting using a microsyringe. Furthermore, when administering to the hippocampus, one to several holes may be made in each cerebral hemisphere, and the conjugate may be injected through these holes in the same manner as described above.

In the case of rats, the administration site to the cerebral cortex is, for example, as described by Faxinos and Watson (
The range of the ``norontal cortex, varietal cortex, and occivital cortex'' in ``The Rat Brain in Stereotaxic Coordination'' Academic Press Sydney (1986), and The site of administration to the hippocampus is C.
It may be selected as appropriate within the range of AI, CAz, and CA3.

The dosage of the conjugate varies slightly depending on the type of animal, but
In general, it is sufficient that the amount is in the range of about 0.5 to 30 μg per individual. For example, when administering to the cerebral cortex of a rat or mouse, it is recommended to administer about 0.5 to 6 μg per rat or mouse. Appropriate. The degree of destruction of cholinergic neurons can be adjusted by appropriately increasing or decreasing the number of mice administered within this range. After administration, the experimental animals of the present invention can be obtained by normal breeding for about 3 to 7 days. can.

Note that the experimental animal of the present invention can also be produced by directly administering a conjugate of NGF and neuronal cytotoxin to Meynert's nucleus, basal forebrain, and/or septal nucleus.

The mammal of the present invention thus obtained has cholinergic neurons that project from the Mynell 1 nucleus or the basal forebrain to the cerebral cortex and/or cholinergic neurons that project from the septal nucleus to the hippocampus. Because it is selectively destroyed,
It can be used as an experimental model of Alzheimer's type dementia or as an experimental animal to investigate the function of basal forebrain and septal nucleus cholinergic neurons.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (1) Preparation of conjugate of NGF and neuronal cytotoxin 2.5S-
NGF (Collaboration/Manufactured by Etive Research) 10μg
0. (Dissolved in 40 μl of 12M phosphate buffered saline and added diphtheria l.
(manufactured by Wako Pure Chemical Industries) 22.3μR to 0. (Add a solution dissolved in 40 μl of 12H phosphate buffered saline. Then,
Add to this mixture a distilled aqueous solution of 1-edyl-3-(3-1,-dimethylaminopropyl)carbodiimide hydrochloride (
Add 80 μl of 100 mg/IIdl) and let stand at room temperature for IO hours while stirring occasionally.

Then, the reaction solution was reduced to 0. (dialyzed overnight at 4°C in 12M phosphate-buffered saline. Thus, 2.5S.NGF-
A dialysate containing a diphtheria toxin conjugate is obtained. A portion of this dialysate was taken and subjected to SOS-polyacrylamide gel electrophoresis, which revealed that there was a band of the conjugate (molecule 1, around 71,000), a band of the A chain of diphtheria toxin (molecule i1, around 21,000), and a band of the B chain of diphtheria toxin. It was confirmed that there was no band (around molecule 137,000).

(2) Administration of conjugate A rat (Wista strain, male, weighing approximately 150 g) was fixed in a stereotaxic apparatus (manufactured by Orimo Kagaku) under anesthesia with bentobarbital sodium (40 μg/kg intraperitoneally administered). Next, the skin on the top of the head is incised along the midline to expose the skull, and the skull is opened 2.5 mm anterior and 2.5 mm lateral to the cruciate suture.
5 open, ■ Rear 0.5 mm, Side 2.5 ram, ■ Rear 3
．． A 5 mm, 3.0 lateral opening is drilled into the skull on both sides.

The dialysate containing the 2.5S·NGF-diphtheria toxin conjugate obtained in (1) above was taken into a microsyringe, and the dialysis solution was poured into the cerebral cortex at a depth of 0. Inject to lpl into 5011l.

After administration, rats are kept under normal conditions for 3 days to obtain rats in which cholinergic neurons projecting from the basal forebrain to the cerebral cortex are selectively destroyed.

(3) Observation of C h A T-positive cells The rats obtained above were treated with bentobarbital sodium (40 μ/
kg intraperitoneal administration) Under anesthesia, the chest was opened, a catheter was inserted into the left aorta through the left ventricle, and 100ml of physiological saline was added, followed by Zamboni? ffl (mixture of 4% rose formaldehyde and 0.2z bicric acid) 5
After perfusion with 00d, the brain is removed and fixed by immersion in Zamboni solution overnight.

The obtained brain specimen was immersed in 3 (12M) sucrose solution for a day and night, then quickly frozen on dry ice, cut into 30 μm thick sections using a cryostat (Moriyasu Seisakusho), and The brain sections were immersed in an anti-choline acetyltransferase antibody solution (0.5 μg/rrdl, rat mouse, manufactured by Boehringer Mannheim) overnight, and then analyzed by indirect fluorescent antibody method. Under the microscope, choline acetyltransferase (C) was detected as an indicator of cholinergic neurons.
h/IT ) positive cells were examined.

As a result, a selective decrease in cholinergic neurons in the basal forebrain on both sides that project to the cerebral cortex (a decrease in choline acetyltransferase-positive cells) and a decrease in immunoreactivity were observed.

Example 2 Rats (Wistar strain,
The skull of a male male, weighing approximately 150 g) was exposed and placed 5. 5mm, lateral 4. Puncture the skull on both sides of the O am. 2.5S・obtained in Example 1-(1)
The dialysate containing the NGF-diphtheria toxin conjugate is taken into a microsyringe and 1 μl is injected into the cerebral cortex at a depth of 4 mm. After administration, rats are raised normally for 3 days to obtain rats in which cholinergic neurons projecting from the septal nucleus to the hippocampus are selectively destroyed. Experimental Example 1 The same procedure as in Example 1 was carried out except that 0.5 μl of the 2.5S NGF-diphtheria toxin conjugate solution was injected only into the cerebral cortex of a rat. The positive minutiae were examined. The results are shown in Figure 1.

The photographs in Figure 1 (A and C are the administered side, B and D are the non-administered side) show that choline acetyltransferase-positive cells and immunoreactivity on the administered side have significantly decreased compared to those on the non-administered side. This shows that administration of the NGF-diphtheria toxin conjugate selectively destroys cholinergic neurons that project from the basal forebrain to the cerebral cortex.

Experimental Example 2 Experimental Purpose> Using mice administered with 2.5S・NGF-diphtheria toxin conjugate solution, a memory acquisition test was conducted on the 3rd day after administration, and a memory retention test was further conducted on the 8th day. , 2.5S
・By administration of NGF-diphtheria toxin conjugate,
We investigated how memory acquisition and memory retention abilities are inhibited.

Experimental Method> ■ Groups of 29 6-week-old S1e:ddY mice weighing 26-30 g, and the administration group had approximately 1.5 ram lateral areas of the frontal cortex and parietal cortex on both sides of the head. , depth 0
63-0.5, 0.2 μl of 2.5S NGF-diphtheria 1 hexine conjugate solution was administered under ether anesthesia using a Hamill 1 syringe. In addition, the same volume of physiological saline was administered to the control group in the same manner as above. ■ On the third day after administration, place the mouse gently in the light chamber of a stethops-through type device, which consists of two light and dark chambers with a passage in the center, with the tail facing the passage. When the animals entered the room, an electric shock (alternating current, 0.5 mA, 3 seconds) was given using an electric shocker/scrambler (RBS, LVE-11') to make them acquire the memory of avoiding the dark room.

■ It was determined that avoidance memory had been acquired if the subject remained in the bright room for 120 consecutive seconds or more, and the degree of memory acquisition was examined using the number of times of shock exposure until memory acquisition as an index.

Results〉 The results are shown in Table 1 below.

Table 1 From the table above, it can be seen that the mice administered the 2.5S・NGF-diphtheria 1・xin conjugate received more shocks than the control group before memory acquisition, and their memory acquisition ability was impaired. I understand.

(2) Recording and transporting the animal that has acquired memory in the memory acquisition test in (1) above. On the 8th day, as above, place the animal in the bright room of the step-through device with its tail facing the passageway. Place the marker on the first lumbar vertebra (L,) of the animal and wait 300 seconds for the marker and both hind legs to completely enter the dark room. Using the number of individuals that required the above as an index, the degree of retention of memories acquired 8 days earlier was examined.

<Keika> The results are shown in Table 2 below.

Table 2 (a) Table 2 (b) From the above table, it can be seen that the average time required for the mice treated with the 2.5S・NGF-diphtheria 1・xin conjugate to enter the dark room was significantly shorter than that of the control group. , poorer retention of darkroom avoidance memory acquired 8 sips ago compared to the control group;
In other words, it can be seen that memory retention ability is impaired.

In addition, the number of mice that took more than 300 seconds to enter the sunny room was significantly lower in the treated group than in the control group (2.5S・N).
Impairment of memory retention ability due to GF-diphtheria toxin conjugate can be seen.

Therefore, it is clear that the 2.5S NGF-diphtheria toxin conjugate impairs both memory acquisition and memory retention abilities in mice.

Experimental Example 3 (No practical purpose) A 2.5S-NGF-diphtheria 1-xin conjugate solution was administered to mice immediately after memory acquisition, and a memory retention test was conducted on the 8th day. We investigated how the ability to retain acquired memories is impaired by administration of a toxin conjugate solution. <Experimental Method> In a memory acquisition test conducted in the same manner as Experimental Example 2,
Mouse determined to have acquired darkroom avoidance memory (power outage 26)
-30 g, 6 weeks old S le:ddY strain) in 1 group 26
Immediately after memory acquisition, the administration group received 2 mice as in Experiment 2.
．． A 5S NGF-diphtheria toxin conjugate solution was administered to the mice, and a control group received physiological saline.

Then, on the 8th day, a memory retention test was conducted in the same manner as in Experimental Example 2 to examine whether the memories acquired 8 days earlier were retained.

<Iro> The results are shown in Table 3 below.

Table 3 (a) Table 3 (b) From the above table, it can be seen that the mice treated with the 2.55-NGF-diphtheria toxin conjugate had a significantly shorter average time to enter the dark room than the control group; It can be seen that the retention of memory for darkroom avoidance acquired a day earlier was inferior compared to the control group, that is, the memory retention ability was impaired.

Additionally, the number of mice that took more than 300 seconds to enter the dark room was significantly lower in the treated group than in the control group.
- Impairment of memory retention ability due to NGF-diphtheria toxin conjugate can be seen.

Therefore, it is clear that the 2.5S.NGF-diphtheria toxin conjugate impairs the memory retention ability of mice once acquired.

Experimental Example 4 Experimental method for co-1 acetyl sulfase
There were 1 group of 7 animals, and for the administration group, a 2.5S NGF-diphtheria toxin conjugate solution prepared in the same manner as in Example 1 was applied to the frontal cortex and parietal cortex on both sides of the head, approximately 1.51 m lateral.
Bentobarbital sodium (40 mg/kg, intracavitary administration III) was administered under anesthesia using a Hamilton syringe to a depth of 0.3-0.5 mm at a dose of 0.2 uf.

In addition, physiological saline was administered to the control group in the same manner.

After being kept normally for 5 days after administration, the animals were decapitated, the cerebrum was removed, and the parietal cortex and basal forebrain were immediately collected after weight measurement. The tissue was homogenized in 20 times the volume of 100 mM Tris-EDTA solution (pH 7.4), of which 10 μl was used for measuring choline acetyltransferase activity and the remaining part was used for protein quantification. Choline acetyltransferase activity is determined by Fonum.
) method [Journal of Neurochemistry (J. Neurochem.) Vol. 24, 407-
409 (1975)), and the protein concentration was measured using a protein assay reagent (manufactured by Japan Bioland Laboratory), and the activity per protein was calculated.

<result> The results are shown in Table 4 below.

From the above table, it is clear that the choline acetyltransferase activity in the cerebral cortex and basal forebrain of the administered group was reduced by about 10χ and 9z compared to the control group.

[Brief explanation of drawings]

Figure 1 shows that in Experimental Example 1, 2.5S·'NGF-
Micrographs of basal forebrain sections (biological form) of rats administered with diphtheria toxin conjugate solution; A, C
indicates the administration side, and B and D indicate the non-administration side. A(X i O O) Ill (X I Q (J), 8+

Claims (14)

[Claims] (1) A non-human mammal in which cholinergic neurons projecting from Meynert's nucleus or the basal forebrain to the cerebral cortex and/or cholinergic neurons projecting from the septal nucleus to the hippocampus have been selectively destroyed. (2) The animal according to claim 1, wherein the mammal is a rodent, a dog, or a cat. (3) The animal according to claim 2, wherein the rodent is a rat. (4) Rats with selective destruction of cholinergic neurons that project to the cerebral cortex. (5) Rats in which cholinergic neurons projecting to the hippocampus were selectively destroyed. (6) By administering a conjugate of nerve growth factor and neuronal cytotoxin to the cerebral cortex and/or hippocampus of a non-human mammal, cholinergic neurons or/ and a method for producing an animal model, which comprises selectively destroying cholinergic neurons that project from the septal nucleus to the hippocampus. (7) The method according to claim 6, wherein the mammal is a rodent, dog, or cat. (8) The method according to claim 7, wherein the rodent is a rat. (9) The method according to claim 6, wherein the nerve growth factor is 2.5S*NGF, β-NGF, or 7S*NGF. (10) The method according to claim 6, wherein the neurocytotoxin is diphtheria toxin, cholera toxin, or ricin. (11) The method according to claim 8, wherein the conjugate is a conjugate of 2.5S·NGF and diphtheria toxin. (12) An animal model characterized by administering a conjugate of nerve growth factor and diphtheria toxin to the cerebral cortex of rats to selectively destroy cholinergic neurons that project from the basal forebrain to the cerebral cortex. How to make (13) A method for producing a model animal, which comprises administering a conjugate of nerve growth factor and diphtheria toxin to the hippocampus of a rat to selectively destroy cholinergic neurons that project from the septal nucleus to the hippocampus. . (14) Conjugate of nerve growth factor and diphtheria toxin.