JP2002112664A - Method for making model animal of glaucoma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for making a glaucoma model in a short time having an intraocular pressure surely increased for a long period of time without requiring a specific technique. SOLUTION: This method for making a model anima of glaucoma is characterized in that a solution having fluidity in injection but loses fluidity in an animal anterior chamber is injected through a needle into an anterior chamber to increase an intraocular pressure.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for developing a therapeutic agent for glaucoma or a method for producing a model animal used for researching glaucoma.

[0002]

2. Description of the Related Art Glaucoma has a very large number of patients and is a serious disease that leads to blindness if its symptoms progress. Much research has been done for that, and much effort has been put into treating it. In that case, it is necessary to create a glaucoma model animal, but this technique will increase intraocular pressure. However, the creation of the model animal is extremely troublesome, and only uncertain methods are known. For example, place a saline solution higher than the animal,
Akaike et al.'S method is known in which a physiological saline solution that falls from there is introduced into an anterior chamber of an animal through an extremely fine glass tube to increase intraocular pressure. According to the method of Ueda et al., After inking ink into the anterior chamber of a rabbit, the ink is accumulated in the outlet of the aqueous humor over a week and the outlet is marked. A method of creating a model by irradiating a laser to the marked portion to close the outlet is known.

However, in these models, for example, according to the method of Akaike et al. Described above, advanced techniques are required for producing a very thin glass tube, and the rise in intraocular pressure is extremely high. It is an internal ischemia model. Further, according to the method of Ueda et al., The ratio of animals that can increase intraocular pressure can not be said to be high, and it is necessary to prepare many animals. Furthermore, there was a problem that it took more than one week to create a model, which was a problem in conducting glaucoma research.
Furthermore, even if a model can be created, there still remains a problem that the period during which intraocular pressure is rising is short.

[0004]

The present inventor has conducted various studies on the creation of a glaucoma model animal in which intraocular pressure has been reliably increased over a long period of time without requiring special techniques in a short period of time. The present invention has been completed, and an object of the present invention is to produce a glaucoma model animal in which intraocular pressure has been reliably increased over a long period of time without requiring special techniques.

[0005]

SUMMARY OF THE INVENTION The gist of the present invention is to inject a solution into an anterior chamber of an animal, which has fluidity at the time of injection but loses fluidity in the anterior chamber, through an injection needle. This is a method for producing a glaucoma model animal characterized by increasing intraocular pressure. That is, the present invention has a fluidity that can be injected from an injection needle before injection into the anterior chamber of an animal, and injects a solution that loses fluidity by being injected into the anterior chamber into the anterior chamber, thereby producing aqueous humor. The glaucoma model animal is created by blocking the flow path in the eye and the discharge path to the outside of the eye, thereby increasing the intraocular pressure.

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.
As the animals used in the present invention, animals commonly used in animal experiments such as rats, rabbits, cats and dogs can be used, but rabbits are particularly preferred. In the present invention, it is necessary that the solution to be injected into the anterior chamber of these animals can be injected into the cornea or conjunctiva through an injection needle. The injection needle used is preferably as thin as possible. If it is thick, there is a problem that the material injected into the anterior chamber leaks from the hole through the injection needle or damages the animal. Specifically, a needle finer than 17G, and more preferably, a needle finer than 24G. Although the amount to be injected differs depending on the animal used, the total amount of the aqueous humor of the animal used in any animal is injected. Specifically, it is about 0.05 mL for rats and about 0.2-0.3 mL for rabbits. It is necessary that the solution to be injected has fluidity that can be injected at the time of injection, loses fluidity in the anterior chamber, and has an excellent affinity for surrounding tissues. Specifically, collagen or a temperature-sensitive synthetic polymer is desirable.

[0007] Collagen is a protein that exists in large amounts in the body of mammals, fish, and the like. It is. As the type of collagen, natural collagen such as acid-soluble collagen, salt-soluble collagen, and solubilized collagen such as enzyme-soluble collagen, alkali-soluble collagen, and chemically modified collagen can be used. In particular, atelocollagen, which is obtained by treating a telopeptide having the antigenicity of a collagen molecule with pepsin to suppress the antigenicity and gelling by applying a certain temperature, is preferable. As raw materials for collagen, animals, fish, etc. can be used, and collagen extracted from tissues containing these collagens, or recombinant collagen produced from cells, fungi, plants, or animal tissues into which genes have been introduced may also be used. it can. As disclosed in JP-A-3-292882, a temperature-sensitive synthetic polymer is also used as a substrate for cell culture, and has excellent affinity with cells. It is preferable that the temperature-sensitive synthetic polymer solution is a neutral solution containing polyisopropylacrylamide.
A solution is prepared so that it can be injected with any of the materials.
Is desirable. The osmotic pressure of this solution is desirably the same as the osmotic pressure of a living body, specifically, 400 to 600 Osm. In order to obtain such a solution, the pH and the osmotic pressure are adjusted by adding a buffer or a salt to the buffer.

[0008] The injected liquid material loses fluidity in the anterior chamber even if it is made of either collagen or a temperature-sensitive synthetic polymer. In the case of collagen, the collagen that has been molecularly dispersed in the solution is heated to form a higher-order structure, thereby gelling and losing fluidity. On the other hand, in the case of a temperature-sensitive synthetic polymer, the polymer forms a hydrate below the lower critical solution temperature (LCST), but when heated to above the LCST, water is released to the outside. The polymers lose their fluidity by coagulation shrinkage and gelation. In the case of the temperature-sensitive synthetic polymer used in the present invention, the lower critical solution temperature (L
When CST) is 30 ° C. or lower, injection into the anterior chamber is possible, and after injection, the gel gels due to body temperature and can be used to create a glaucoma model. Specifically, in the case of collagen, it becomes cloudy after injection of the anterior chamber, and the intraocular pressure increases. On the other hand, in the case of a temperature-sensitive synthetic polymer, the intraocular pressure increases while maintaining transparency.

[0009]

EXAMPLES The present invention will be described in detail below with reference to examples, but the technical scope of the present invention is not limited by these examples. Example 1 0.1 M of enzyme-solubilized collagen (atelocollagen)
Prepare a phosphate buffer (pH 7.2). This solution is a solution that maintains transparency at low temperatures. When 0.2 mL of this was injected into the anterior chamber of the rabbit, intraocular pressure increased 24 hours later. This increase in intraocular pressure persisted for 3 weeks after anterior chamber injection.

Example 2 When 0.05 mL of the enzyme-solubilized collagen solution used in Example 1 was injected into the anterior chamber of a rat, intraocular pressure increased after 24 hours. This increase in intraocular pressure persisted for two weeks after anterior chamber injection. Example 3 Enzyme-solubilized collagen (atelocollagen) was added to 0.02
Prepare M phosphate buffer + NaCl (pH 7.2). When 0.2 mL was injected into the rabbit anterior chamber in the same manner as in Example 1, 24
The intraocular pressure increased after hr. This increase in intraocular pressure persisted for 3 weeks after anterior chamber injection.

Example 4 An N-isopropylacrylamide monomer is dissolved in benzene and polymerization is carried out with 2,2-azibisisobutyronitrile. The polymer precipitated at 60 ° C. for 12 hours under a nitrogen stream was collected and purified by precipitation with ethyl ether. A 5% aqueous solution of the obtained poly-N-isopropylacrylamide was prepared, and was added to the rabbit anterior chamber in the same manner as in Example 1.
When 2 mL was injected, intraocular pressure increased 24 hours later.
This increase in intraocular pressure persisted for one week after anterior chamber injection.

[0012]

As described above, a solution that has fluidity during injection but loses fluidity in the anterior chamber is used.
By injecting into the anterior chamber of an animal, intraocular pressure can be increased and the increase in intraocular pressure can be maintained for a long period of time, so that it is extremely suitable as a glaucoma model animal.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takayuki Kanda 2-1-1 Hongo, Bunkyo-ku, Tokyo Juntendo University Ophthalmology Classroom (72) Inventor Nobuhiko Tachibana 2-1-1 Hongo, Bunkyo-ku, Tokyo Juntendo University Eye Inside the Science Classroom (72) Inventor Shinji Nakamura 2-1-1 Hongo, Bunkyo-ku, Tokyo Juntendo University Ophthalmology Classroom (72) Inventor Yasuo Watanabe 2-1-1 Hongo, Bunkyo-ku, Tokyo Juntendo University Ophthalmology Classroom ( 72) Inventor Atsushi Kanai 2-1-1 Hongo, Bunkyo-ku, Tokyo F-term in the Department of Ophthalmology, Juntendo University 2G045 AA29 AA40 BB60 CB17 DA77

Claims (6)

[Claims] 1. An intraocular pressure is increased by injecting a solution having fluidity at the time of injection but losing fluidity in the anterior chamber through an injection needle into an anterior chamber of an animal. How to make a glaucoma model animal. 2. The method for producing a glaucoma model animal according to claim 1, wherein the solution losing fluidity in the anterior chamber is a collagen solution. 3. The method according to claim 2, wherein the collagen solution is a neutral solution of enzyme-solubilized collagen. 4. The method for producing a glaucoma model animal according to claim 1, wherein the solution that loses fluidity in the anterior chamber is a temperature-sensitive polymer solution. 5. The glaucoma model animal according to claim 4, wherein the temperature-sensitive polymer solution is a neutral solution of a temperature-sensitive polymer having a lower critical solution temperature of 30 ° C. or less. How to make. 6. The glaucoma according to claim 5, wherein the temperature-sensitive polymer having a lower critical solution temperature of 30 ° C. or lower is a temperature-sensitive polymer containing at least polyisopropylacrylamide. How to create a model animal.