WO2004049793A1

WO2004049793A1 - Model animal of cognitive learning disability with the use of leukemia inhibitory factor and method of constructing the same

Info

Publication number: WO2004049793A1
Application number: PCT/JP2003/015037
Authority: WO
Inventors: Hiroyuki Nawa
Original assignee: National University Corporation Niigata University
Priority date: 2002-11-29
Filing date: 2003-11-25
Publication date: 2004-06-17
Also published as: JP2004180655A; AU2003284688A1

Abstract

It is intended to provide an animal showing cognitive learning disability which comprises the step of administering a specific protein factor disturbing the brain function development of the animal or disrupting the developed brain structure of the animal or expressing a gene of the protein factor to achieve the same effect as the administration; and a means of evaluating a remedy for mental diseases associated with cognitive learning disability such as autism and schizophrenia or evaluating the availability of a remedy therefor.

Description

BACKGROUND OF THE INVENTION Model animal of cognitive learning disorder using leukemia inhibitory factor and its production method

1. Field of the technology

The present invention relates to a method for preparing a brain disease model animal and its application in the fields of medicine and pharmacy.

It is about new technology.

2. Background technology

book

Many mental illnesses still have no apparent biological etiology or pathology, and many patients suffer from intractable diseases. For example, autism occurs in 1 in 500-250 people, and is still effective for mental illness represented by cognitive impairment such as social repulsion and mental retardation such as learning disability. There is no. Schizophrenia also affects 0.7-1.0% of the population, producing hundreds of thousands of long-term hospitalized patients in Japan. The main symptoms of this disease are positive symptoms such as delusions, hallucinations, and hallucinations, as well as cognitive deficits such as paresthesias and various mental abnormalities, such as bowel I withdrawal and depression. It accompanies. At present, the elucidation of the etiology is not yet clear. Thus, much is unknown about basic medicine for mental illness with cognitive learning disabilities.

Schizophrenia develops from adolescence to middle age with symptoms characteristic of perception, thinking, emotion, and behavior, and is often a chronic disorder that causes various difficulties in social adjustment. There are two types of mental symptoms: positive symptoms (such as hallucinations, delusions, weakening thoughts, nervous symptoms, and strange behavior) and negative symptoms (such as flattened emotions, reduced motivation, and social withdrawal). It is hoped that the social ladle will establish an integrated and comprehensive treatment system, including early detection, treatment, social rehabilitation activities, and prevention of recurrence, based on the specific nature of the disease. The only therapeutic agent that improves the positive symptoms of schizophrenia is the neurotransmitter dopami Drugs that antagonize tongue mouth tonin are said to be useful. In many cases, long-term administration of these drugs over the years is indispensable. Specifically, phenothiazine compounds, thioxanthine compounds, petit mouth phenone compounds, and benzamide compounds are frequently used in patients.

In addition to studying the mechanism of action of these drugs, the hypothesis that methamphetamine represented by amphetamine actually induces positive symptoms of schizophrenia in humans, and that dysfunction of dopamine causes schizophrenia. Has been advocated. Based on these facts, animals receiving chronic administration of amphetamine may be used as a model for schizophrenia. Similarly, drugs that have hallucinogenic effects in humans may be used as animal models for schizophrenia when administered to animals. For example, animals administered fencitaridine, which is an inhibitor of glutamate receptor, which is said to be related to brain physiology of memory and learning, is that.

Many of these conventional animal models of schizophrenia exhibit a transient cerebral dysfunction dependent on drug administration, and do not necessarily reproduce the pathology of chronic schizophrenia as seen in humans. I couldn't say. In addition, the action of dopamine antagonists was mainly for the improvement of positive symptoms, and therapeutic drugs for negative symptoms were extremely limited. This may be due to the lack of a suitable animal model for schizophrenia.

Until now, various hypotheses have been proposed for the pathogenesis of schizophrenia and autism. One of them is the hypothesis of neurodevelopmental disorders proposed by researchers mainly by Winberger (Reference 1). However, what biological factors actually cause abnormalities in cranial nerve development by what mechanisms. It was completely unknown what caused it. The present invention provides an animal model that scientifically embodies the hypothesis. Although a cognitive impairment model has been reported based on this hypothesis by administration of growth factors and cytodynamics, the animal model according to the present invention has a decrease in locomotor activity similar to withdrawal behavior and a learning disability. Has the characteristic that it occurs simultaneously (Patent applications 1, 2, Literature 8).

On the other hand, autism is more hereditary than schizophrenia, but its cause is still largely unknown. In animals, it has been reported that repeated stress on the fetal mother causes similar cognitive learning disorders in the offspring. Recently, abnormal development of serotonergic nerves in the brainstem has been discussed (Reference 2). In this sense, as in schizophrenia, the hypothesis that developmental dysfunction of cranial nerves is influential is strong, but there are few effective therapeutic agents (Reference 3).

Summary of the Invention

An object of the present invention is to provide a method for producing an animal model showing a cognitive learning abnormality and a method for using the same.

In order to solve the above-mentioned problems, the present invention relates to a step of over-administering a specific protein factor involved in brain function development or controlling post-development brain cell function, or overexpressing the gene thereof. The method comprises:

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail, but the description and examples of these preferred embodiments do not imply any limitation or control of the effective range of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing changes in prepulse inhibition with growth of rats in which cognitive behavioral abnormalities were induced by administering leukemia inhibitory factor (LIF) during infancy.

FIG. 2 is a graph showing the time course of waterfront activity at the age of three weeks after birth in rats to which leukemia inhibitory factor (LIF) was administered during infancy.

FIG. 3 is a graph showing startle response and prepulse inhibition caused by intracerebral administration of leukemia inhibitory factor (LIF).

FIG. 4 is a graph showing the amount of movement of a rat to which leukemia inhibitory factor (LIF) was administered in the brain in a novel environment.

Figure 5 shows conditioned avoidance learning ability of rats treated with leukemia inhibitory factor (LIF) intracerebrally. FIG.

Detailed invention of preferred embodiment

Generally, any animal species such as monkeys, dogs, cats, cat egrets, guinea pigs, and rats can be used with any of the mammals. Usually, rats and monkeys, which have accumulated a large amount of data so far, are considered optimal.

Leukeiraia Inhibitory Factor is positioned as an inflammatory cytokine, but is known to play a major role in the development and differentiation processes including the brain. Cholinergic neurons are known for their academic and historical background. It has many other names such as differentiation factor (Cholinergic Differentiation Factor), D factor, Differentiation-Stimulating Factor). In addition, there are oncostatin M and ciliary neurotrophic factor as related cytokines that bind to similar molecular receptor complexes, and these are used in place of leukemia inhibitory factors, and the cognitive learning described in the claim is used. It is also possible to produce a disabled animal.

There are roughly two methods for administering or expressing these protein factors. One method is to inject these protein factors directly into the abdominal cavity, subcutaneously, or into the brain of animals. According to this method, the concentration of the protein factor in the body is immediately reduced due to metabolic degradation of the protein factor, so it is necessary to inject several times over several times. In addition, in the case of administration from the periphery, the site of action is the brain, so it is ideal to administer immature infants at the blood-brain barrier. In general, these protein factors may be those produced in large amounts in bacteria by genetic recombination or those purified from animal cells.

An appropriate dose of leukemia inhibitory factor is 0.25 mg gZl / kg body weight per animal for systemic administration, or 0.2 SmgZl / kg brain weight / kg for intracerebral administration. But it may be more. However, it should be noted that excessive administration of leukemia-inhibiting factors causes excessive weight loss because they have a weight loss effect. One dose is not enough, preferably 3 It is desirable to have the number of times or between 30 and more times and days.

Another method is to introduce a gene for leukemia inhibitory factor or an analogous protein factor into an individual animal by a recent embryo manipulation method and express the gene in one of the organs including the brain. Is achieved. According to this method, once the transgenic animal is produced, it can be used as the model animal as it is simply by maintaining the strain. For the same purpose, the intracellular signal transduction molecule, which is originally known to activate the protein factor specified in the cell, can be used for genetic manipulation, etc., as if the protein factor acted on the cell. It is also possible to produce a mutant-modified animal using developmental engineering and substitute it.

Similarly, by cloning the leukemia inhibitory factor and analogous protein factor genes into an appropriate gene expression vector, and transfecting and expressing them in the brain of a postnatal animal, the resulting protein factor is consequently expressed. It can also produce the same effect as administration. Examples of gene expression vectors that can be used include retrovirus vectors, adenovirus vectors, henorevirus vectors, and the like.

Animal models of cognitive learning created by this method will exhibit cognitive behavioral abnormalities and learning disabilities as seen in autistic and schizophrenic patients. The abnormal cognitive behavior can be evaluated by the following method. First, behavioral measurements such as prepanolase inhibition of startle response, cognitive performance in various learning tasks, and animal locomotion correspond to this.

Prepulse inhibition in startle response is a test of sensory-motor response ability using the startle response as an index that can be commonly evaluated in humans and animals. In this test, the abnormalities in attention and brain information processing, which are considered to be central to the pathology of schizophrenia and autism, have characteristics that can be evaluated scientifically and objectively. Before the test itself snarls with a loud sound of about 120 dB and causes a startle response, in 30-150 ms, the weak sound stimulus that itself cannot swell and cause a startle response (prepulse) Pre-listening to) measures the startle response with a loud sound. The decrease due to this prepulse is called prepulse inhibition, and it is known that it shows an abnormal decrease in schizophrenia patients and autism patients (Reference 4).

The ability to perform various learning tasks in animals can be measured, for example, in Pavlov-type conditioning learning, such as learning of electric shock and avoidance behavior, spatial learning in the water maze, and bait position learning in the 8-way maze. (Reference 5).

Here, we consider that the animal momentum reflects the phenomenon that autistic patients and schizophrenic patients with negative symptoms reluctantly come into contact with the new environment and withdraw from society. In general, animals measure exploratory activity in a new and stressful environment. Generally, in animal models, this index is estimated to decrease.

References

(Reference 1) Weinberger DR. Arch. Gen Psychiatry 44: 660-669 (1987)

(Reference 2) Hornig M, Lipkin WI.Ment Retard Dev Disabil Res Rev.

7 (3): 200-210 (2001)

(Reference 3) Whitaker-Azmitia PM.Brain Res Bull.; 56 (5): 479-485 (2001) (Reference 4) Braff DL, Geyer MA.Arch Gen Psychiatry 47: 181-188 (1990) (Reference 5) Sams-Dodd F. Rev Neurosci. 10 (1): 59-90. (1999)

Related prior inventions and research by the applicant include:

(Patent application 1) 2000/10/10 application; Japanese Patent Application 2000-309042 Schizophrenia-like abnormal cognitive behavior and its production

(Patent application 2) Filed February 27, 2001; Japanese Patent Application 2001-52546 Animal model for schizophrenia-like psychiatric disorder, its production method and its use

(Bunnan Inu 6) Nawa H, Takahashi M, Patterson PH. Mol Psychiatry.

Nov; 5 (6): 594-603 (2000)

(Reference 7) Takahashi M, Shirakawa 0, Toyooka K, Kitamura N, Hashimoto T, Maeda K, Koizumi S, Wakabayashi H, Someya T, Nawa H. Mol Psychiatry. May; 5 (3): 293-300 (2000)

(Document 8): Futamura T, Toyooka K, Iritani S, Niizato K, Nakamura R, Tsuchiya K, Someya T, Kakita A, Takahashi H, Nawa H. Mol Psychiatry. 7 (7): 673-682. (2002 )

According to the principle and method of the present invention, an animal model of autism, schizophrenia, and similar cognitive learning disorders can be easily provided. In addition to using the drug as a model animal, it will be possible to develop and evaluate therapeutics and diagnostics for autism ゃ schizophrenia and similar mental disorders with cognitive learning disorders.

Example

First, an example of peripheral administration of Leukeimia Inhibitory Factor to L children will be described.

Example 1: Abnormal startle prepulse inhibition caused by administration of Leukeimia Inhibitory Factor to infants in rats

The animals were used from the age of 2 days after birth of SD rats (Japan SLC). As reagents, mouse recombinant leukemia inhibitory factor (GIBCO Lifetech) and cytochrome C (Sigma) as a control were dissolved in physiological saline. From the second day after birth, a total of 10 times every other day (until the 11th day after birth) was administered subcutaneously to the cervix of 0.25 microdalum per gram of rat body weight. From 3 weeks after birth, startle response intensity and prepulse inhibition were measured using a small animal startle response measuring device (San Diego Instruments). That is, a sound stimulus (120 dB) is used as a sensory stimulus to induce a startle response, and a stimulus (75 dB) that is 6 dB higher than the environmental noise (background noise) level is used as a prepulse stimulus. ), And after 100 milliseconds, a pulse stimulus with a sound pressure of 120 dB was given. The response ratio of the startle response when 120 dB alone and the prepulse were combined was defined as prepulse inhibition (PPI). From 3 weeks to 8 weeks after birth, the leukemia inhibitory factor group showed a decrease in the ANOVA test, especially at the age of 6 weeks after birth, the leukemia inhibitory factor group alone compared to the cytochrome C group. Prepulse inhibition was significantly reduced (* P <0.05) (Fig. 1). In FIG. 1, open circles indicate healthy control rats, solid circles indicate rats treated with leukemia inhibitory factor (LIF), # * indicates a significant change point, and n indicates the number of animals examined. The vertical axis is the prepulse inhibition (%), and the horizontal axis is the age after birth. '

Example 2: Novel abnormal environmental locomotion caused by infant administration of leukemia inhibitor factor

The animals were used from the age of 2 days after birth of SD rats (Japan SLC). As reagents, mouse recombinant leukemia inhibitory factor (GIBCQ Lifetech) and cytochrome-I C (Sigma) were dissolved in physiological saline. From the second day after birth, 0.25 micrograms per gram of rat body weight was subcutaneously administered subcutaneously to the neck 10 times every other day (until the 11th day after birth). The activity of the animal in the new environment was determined by placing the rat in a new box of about 50 cm square, and then quantifying the amount of exercise using a videotape and an activity analysis system (Neuroscience) linked to the videotape. At 3 weeks of age, the leukemia inhibitory factor group showed a significant decrease in the AN OVA test, especially in the 5-minute period immediately after being placed in the new environment. It showed a significant decrease in momentum (* p 0.05) (Figure 2). In FIG. 2, open circles indicate healthy control rats, solid circles indicate rats administered with leukemia inhibitory factor (LIF), and n indicates the number of animals studied. The vertical axis is the horizontal movement distance (cm), and the horizontal axis is the time (minutes) after placing in the new environment.

Further, an example of intramuscular administration of leukemia inhibitory factor (Leukeimia Inhibitory Factor) to adult rats will be described.

Example 3: Abnormal startle prepulse inhibition caused by intracerebral administration of leukemic inhibitory factor (Leukeimia Inhibitory Factor) The animals were used from the age of 56-66 days after birth of SD rats (Japan SLC). As a reagent, mouse recombinant leukemia inhibitory factor (GIB CO Lifetech) was dissolved in physiological saline. Under anesthesia, the rat was implanted with a 28-gauge cannula 0.5 mm anterior to Bredama, 3.Omm in the hole drilled in the skull with a dental drill on the side of the dentist. did. A plastic tube is connected to the end of the force-Yure carried in the striatum and connected to an osmotic pump (250 microliter volume (AZLET raodel2002, AZLA CORP; continuous administration for 14 days). The osmotic pump was previously filled with mouse recombinant leukemia inhibitory factor (40 micrograms / m1; GI BCO Ranhutech) and saline. After suturing the scalp, I fastened it with a surgical clip and waited for recovery from surgery.

From one week after the start of the administration, the startle response intensity and prepulse inhibition were measured using a small animal startle response measuring device (San Diego Instruments). That is, a sound stimulus is used as the sensory stimulus that induces the startle response, and an environmental noise is

(Background noise) A stimulus with a sound pressure 5 dB higher than the level was applied, and 100 ms later, a pulse stimulus with a sound pressure of 120 dB was applied. The response ratio when the startle response when using 120 dB alone and the prepulse were combined was defined as prepulse inhibition (PPI). Decrease in responsiveness to a 120 dB pulse stimulus before and after these sessions was assessed as adaptability. The leukemia inhibitory factor-treated group showed a significant decrease in all prepulse intensities as compared with the saline-treated group. However, there was no significant difference between the startle response intensity and the degree of acclimation itself when 120 dB was used alone (FIG. 3). In Fig. 3, the upper figure is a graph showing the decrease rate of the main startle response when a sound pre-pulse of 75 dB, 80 dB, and 85 dB is applied, and the lower left figure is a graph of 120 dB without the pre-pulse. Draft showing the primary startle response. The lower right figure is a graph showing the change in the primary startle response at 120 dB before and after the test. In FIG. 3, a white bar represents a control animal to which saline was administered, and a black bar represents a control animal. Bars indicate animals that received leukemia inhibitory factor (LIF) intracerebrally, and P indicates statistical significance. The vertical axis in the upper figure is prepulse inhibition (%), the vertical axis in the lower left figure is 120 dB startle response intensity, and the vertical axis in the lower right figure is 120 d before and after the test. B is the primary startle response rate (%).

Example 4: Leukimia Inhibitory Factor Induced Novel Environmental Exercise by Abdominal Brain Administration

As shown in Example 3, SD rats (Japan SLC) 56-66 days old after birth were injected with a recombinant mouse leukemia inhibitory factor (GI BCO Ranhutech) and saline in an osmotic pump (AZLET model 2002, AZLA CORP). For 14 S. One week after the start of administration, the following exercise test was performed. The amount of exercise in the new environment was determined by placing a rat in a new box of about 50 cm square and then quantifying the amount of exercise using a videotape and an activity analysis system (Neuroscience) linked to it for 30 minutes. The leukemia inhibitory factor group showed a tendency to decrease in horizontal momentum (p = 0.088) and a significant decrease in vertical movement (p = 0.032) in the Student T test compared with the saline-treated group. (Figure 4). In Fig. 4, the left figure shows the horizontal momentum, and the right figure shows the vertical momentum. The white bar indicates a control rat administered with saline, the black bar indicates a rat to which leukemia inhibitory factor (LIF) was intracerebrally administered, n indicates the number of animals examined, and P indicates statistical significance.

Example 5: Decreased ability to learn conditioned avoidance caused by intracerebral administration of Leukeimia Inhibitory Factor

As shown in Example 3, SD rats (Japan SLC) 56-66 days old after birth were injected with a recombinant mouse leukemia inhibitory factor (GI BCO run foottech) and saline in an osmotic pump (AZLET model 2002, AZLA CORP). For 143 days. On the 10th day after the start of the administration, the ability to avoid avoidance learning was measured. At the time of the test (7-9 weeks old), using a two-way condition avoidance behavior device (Muromachi Kagakusha), when the sound and the light of 80 dB flash, it is necessary to move to the next room. If not move, electricity Learn by applying a shock (0.6 mA, 10 seconds). This task was repeated 90 times at 10-50 second intervals (randomly), and the learning ability was judged based on the correct response rate to the conditional stimulus. Regarding continuous administration to the brain of growing rats, the leukemia inhibitory factor group showed a significant decrease in learning ability in almost all learning sessions compared to the physiological saline group (* p <0.05). (Figure 5). In FIG. 5, the solid line “diamond point” indicates a control rat administered with physiological saline, the solid square “dotted line” indicates a rat administered with leukemia inhibitory factor (LIF) intracerebrally, and n indicates the number of animals examined. The vertical axis is the correct answer rate (%), and the horizontal axis is the number of learning sessions (times).

Claims

The scope of the claims

1. A method for producing an animal that induces cognitive learning deficits with persistent cerebral dysfunction by administering a leukemia inhibitory factor (Leukeimia Inhibitory Factor) to a young animal during development.

2. Use of an animal created by the method of claim 1 as a model for cognitive learning disability.

3. The method according to claim 2, wherein the method is used for evaluating a therapeutic agent or a therapeutic agent for autism, schizophrenia or a cognitive learning disorder mental disorder.

4. A method of producing an animal that has induced cognitive learning disorders with acute cerebral dysfunction by administering a leukemic inhibitory factor (Leukeimia Inhibitory Factor) to a developing animal.

5. Use of an animal created by the method of claim 4 as a model for cognitive learning disability.

6. The use according to claim 5, which is intended to evaluate the therapeutic potential of autism, schizophrenia, or cognitive learning disorder mental illness.

7. A method for producing an animal in which a cognitive learning disorder exhibiting cerebral dysfunction is induced by overexpressing a leukemia inhibitory factor (Leukeimia Inhibitory Factor) gene in an animal embryo and forcibly expressing the leukemia inhibitory factor in the animal. .

8. Use of an animal created by the method of claim 7 as a model for cognitive learning disability.

9. The use according to claim 8, which is intended to evaluate the therapeutic potential of autism, schizophrenia or cognitive learning disorder mental illness.

10. A method for producing an animal that induces cognitive learning disorders exhibiting cerebral dysfunction by overexpressing the leukemia inhibitory factor (Leukeimia Inhibitory Factor) gene in postnatal animals and forcibly expressing the leukemia inhibitory factor in the animals. .

11. Use of an animal created by the method of claim 10 as a model for cognitive learning disability.

12. The use according to claim 11, wherein the use is intended to evaluate the therapeutic potential of autism, schizophrenia or cognitive learning impaired mental illness.