JP2003329672A - Apparatus for measuring amount of carbon dioxide generated by respiration of experimental animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To solve the problem with measuring the concentration of carbon dioxide resulting from the respiration of an experimental animal that, while directly measuring the concentration of carbon dioxide produced by the animal requires air to be stably supplied to a sealed breeding cage at a constant rate, controllers currently in use cannot deal with mice, etc. <P>SOLUTION: This apparatus includes three systems ((1) for treated animals; (2) for animals yet to be treated; and (3) for measuring the total amount of carbon dioxide contained in air supplied to the breeding cage) each having a sealable breeding cage 3; a gas supply means 23 or the like for supplying air into the cage 3 at a constant rate; and a collection cylinder 49 or the like. The collection cylinder 49 is filled with soda lime, and CO<SB>2</SB>is converted to Na<SB>2</SB>CO<SB>3</SB>through a chemical reaction and then fixed in the collection cylinder 49. Thus, the weight of CO<SB>2</SB>collected by each of the systems can be determined from a change in the weight of the collection cylinder 49; based on a difference in the weight of CO<SB>2</SB>between system (1) (or (2)) and system (3), the amount of carbon dioxide produced by the respiration of the experimental animal can be quantitatively determined. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal, and more particularly to an experimental animal suitable for use when a very small animal such as a mouse or rat is used as the experimental animal. The present invention relates to a device for measuring the amount of carbon dioxide produced by breathing.

[0002]

2. Description of the Related Art Since it is possible to know the physiological function of an animal by quantifying the respiration rate of the animal, quantifying the respiration rate of the animal is an essential experimental technique in the development of pharmaceuticals. Since human experiments are not possible at present, many laboratory animals are used instead of humans, and these provide various information. Although many animals are used as experimental animals, among them, extremely small animals such as mice and rats that are easy to breed and have a short life cycle are useful.

Since it is extremely difficult to measure the respiration rate itself of a laboratory animal, the amount of carbon dioxide generated by respiration is usually measured. The measuring device currently on the market controls the amount of air supplied to a closed breeding tank at a constant level, measures the amount of carbon dioxide produced by respiration of an experimental animal in the breeding tank in terms of concentration, and uses the following formula (1 ), The amount of carbon dioxide produced by respiration of the experimental animal is calculated. Cw = 44 × (Cc−Cb) × F × 1440/22400 (1) Where, Cw: Carbon dioxide generation by respiration (g / d)
ay) Cc: carbon dioxide concentration in breath (%) Cb: carbon dioxide concentration in air (%) F: amount of air supplied to a closed breeding tank (mL / m
in)

Recently, non-dispersive infrared absorption method (NDIR)
By using such as, carbon dioxide concentration, that is, Cc
There are measuring devices on the market that can measure the On the other hand, in order to accurately measure Cw, it is necessary to stably supply air to the breeding tank with a constant air supply amount for a long period of time. However, the accuracy level of constant flow rate supply by the controller of the current commercially available device is 1 L / m.
It can only handle insufflation rates of in (1,440 L / day) or more. Laboratory animals that can meet such specifications are larger than rabbits and have a respiratory volume of approximately 30 mL /
When a very small animal such as a mouse (min) (44 L / day), rat, etc. is used as an experimental animal, it must be measured as a group of several to several tens.

Therefore, in order to measure the amount of carbon dioxide produced by respiration of a single extremely small animal, a device which uses a substance labeled with a radioisotope and measures the produced radioactive carbon dioxide is used. However, special facilities (R
I facility) and equipment are required, and further the radiation handling chief qualification is required for measurement. In addition, the health of the measurer,
There are serious problems such as the impact on the environment cannot be ignored and special and expensive reagents are required.

[0006]

SUMMARY OF THE INVENTION Therefore, the present invention is an apparatus for solving the above-mentioned problems and capable of accurately measuring the amount of carbon dioxide produced by respiration of an extremely small animal, and there is no restriction on the place of experiment or the person making the measurement. An object of the present invention is to provide a highly convenient measuring device that does not require a special and expensive reagent at the time of measurement.

[0007]

In order to solve the above-mentioned problems, the invention of claim 1 provides a breeding tank capable of being sealed, and a gas supply means for supplying an oxygen-containing gas to the breeding tank at a constant air supply amount. And a series having a collecting means that collects carbon dioxide contained in the exhaust gas from the breeding tank after it has been converted into a form quantifiable by a gravimetric method by a chemical reaction and then collected.
A device for measuring the amount of carbon dioxide produced by respiration of an experimental animal, wherein the amount of carbon dioxide produced by respiration of the experimental animal is calculated based on the weight measurement of the carbon dioxide collected by the collecting means. .

[0008] The invention of claim 2 is an apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to claim 1, wherein an experimental series for raising treated animals for experiment in a breeding tank, and a breeding tank It has three series consisting of a control series for raising untreated animals for a control experiment inside and a reference series for measuring the total amount of carbon dioxide contained in the oxygen-containing gas supplied to the breeding tank. It is a measuring device.

A third aspect of the present invention is the apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to the first or second aspect, further comprising the carbon dioxide contained in the oxygen-containing gas supplied to the breeding tank. A measuring device comprising a carbon dioxide concentration control means for controlling the concentration.

According to a fourth aspect of the present invention, in the apparatus for measuring the amount of carbon dioxide produced by respiration of a laboratory animal according to any one of the first to third aspects, the gas supplied to the breeding tank is further sterilized. The measuring device is provided with a sterilization processing means.

According to a fifth aspect of the present invention, in the apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to any one of the first to fourth aspects, the breeding tank comprises an outer closed container and an inner cage. The measuring device has a layered structure, and the cage is detachably mounted inside the closed container.

The invention of claim 6 is the apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to any one of claims 1 to 5, wherein the breeding tank is made of a heat-resistant material. It is a measuring device.

According to a seventh aspect of the present invention, in the device for measuring the amount of carbon dioxide produced by respiration of a laboratory animal according to any one of the first to sixth aspects, the urine and feces of the laboratory animal are collected in the lower part of the breeding tank. The measuring device is provided with a means.

The invention according to claim 8 is the measuring device for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to any one of claims 1 to 7, which is for a very small animal. is there.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 schematically shows the whole measuring device 1 for measuring the amount of carbon dioxide produced by respiration of an experimental animal, and FIG. 2 schematically shows one series included in the measuring device 1. First, one series shown in FIG. 2 will be described. Reference numeral 3 indicates a breeding tank, in which micro animals such as mice are put and raised as experimental animals. The container body 5 and the lid 7 that closes the opening surface of the upper part thereof form a container that can be sealed. The lower part of the container body 5 has a conical shape that tapers downward, and the lowermost part is connected to one end of the pipe 9.

The other end of the pipe 9 serves as a collection port 11 for collecting urine and feces. A valve 13 is provided in the middle of the pipe 9, and when the valve 13 is opened, urine and feces of an experimental animal are guided to the collection port 11 through the pipe 9. The pipe 9 constitutes a means for collecting urine and feces. By providing such a collecting means, urine and feces can be collected at the same time. Therefore, according to the measuring device 1, in addition to the quantitative analysis data of the carbon dioxide generation amount due to respiration, the quantitative analysis data of the metabolite can be acquired at the same time. In addition, the container body 5 is the support portion 1
Supported by 5.

A net-like cage 17 is arranged in the container body 5. A handle (not shown) is provided on the cage 17 so that the cage 1 can be kept with the experimental animals.
7 can be taken out from the container body 5. Therefore, by weighing the cage 17 with the experimental animal in it, the animal weight can be measured, and it is possible to prevent the worker from being bitten and scratched, and at the same time, there is no need to capture it, so do not chase it. Rearing stress of experimental animals can be relieved.

A heater 19 and a temperature sensor 21 are attached to the lid 7, and the inside of the breeding tank 3 is maintained at a desired constant temperature. The container main body 5, the lid 7 and the cage 17 that constitute the breeding tank 3 are all made of a heat-resistant material such as stainless steel, and when designed for a very small animal, they are small, so It can be easily sterilized in an autoclave (high-pressure steam oven).

Reference numeral 23 indicates a pipe, and this pipe 23
One end of the penetrating into the breeding tank 3 from the lid 7. The other end is
It has entered the series header (container) 25. In the middle of the pipe 23, the gas analysis port 27 and the sterile filter 2
9 is provided. The gas analysis port 27 is provided for precise analysis of the air supplied to the breeding tank 3 for checking the device safety of the measuring device 1, and a gas syringe or the like can be appropriately connected to this before measurement work or the like. Air is collected and the collected air is analyzed by gas chromatography or the like. The aseptic filter 29 is provided to remove various microorganisms from the air supplied to the breeding tank 3.
It constitutes sterilization processing means.

The series header 25 is provided for supplying a test liquid agent, a test reaction liquid, a test gas and the like, and in this embodiment, the test liquid agent L is put therein. If the series header 25 is filled with water instead of the test liquid agent or the like, it can be used as a humidity controller. One end of the pipe 31 is inserted in the series header 25,
The other end of the pipe 31 is connected to the header 33 of the entire measuring device 1. A valve 35 and an integrating flowmeter 37 are provided in the middle of the pipe 31. The valve 35 adjusts the amount of air accumulated in the header 33 supplied to the breeding tank 3. The integrated flow meter 37 is for measuring the total amount of air supplied to the breeding tank 3.

Reference numeral 39 indicates a pipe, and this pipe 39
Is connected to the breeding tank 3, and the air in the breeding tank 3 is discharged from the breeding tank 3 through the pipe 39. A gas analysis port 40 is provided in the middle of the pipe 39. The gas analysis port 40 is provided for precise analysis of the air discharged from the breeding tank 3, and a gas syringe or the like is appropriately connected to this during measurement work to collect air, and the collected air is a gas. Analyze by chromatography method. Reference numeral 41 indicates a gas processing unit. Ammonia absorption bottle 43, first dehumidifying cylinder 45, second dehumidifying cylinder 47, collecting cylinder 49 and moisture absorbing cylinder 51
Are sequentially assembled from the bottom so as to be attachable to and detachable from each other via the connecting portion 53. On the connecting portion 53 arranged at the uppermost portion, a discharge cylinder 55 which is tapered upward is arranged. Ammonia absorption bottle 43, first dehumidifying cylinder 45, second dehumidifying cylinder 47,
Collection cylinder 49, moisture absorption cylinder 51, discharge cylinder 55, connecting portion 53
The gas processing unit 41 is constituted by.

In order to remove ammonia (NH ₃ ) contained in the gas discharged from the breeding tank 3 through the pipe 39, the ammonia absorption bottle 43 contains 1M-H ₂ SO.
₄ is filled. First dehumidifying cylinder 45, second dehumidifying cylinder 47
Is used to remove water (H ₂ O) contained in the gas from which ammonia (NH ₃ ) has been removed.
O ₂ ) and silicon dioxide (SiO ₂ ) and calcium chloride (CaCl ₂ ) are respectively filled.

A collecting cylinder 49 as a collecting means is filled with soda lime (NaOH). Ammonia (N
H ₃ ) and water (H ₂ O) removed gas passes,
Carbon dioxide (CO ₂ ) gas contained in the gas is converted into sodium carbonate (Na ₂ CO ₃ ) by the following chemical reaction, and then fixed on soda lime and collected. 2NaOH + CO ₂ → Na ₂ CO ₃ + H ₂ O

The water absorption cylinder 51 is filled with calcium chloride (CaCl ₂ ) in order to absorb the water (H ₂ O) generated by the above chemical reaction. Water absorption cylinder 51
After the water has been absorbed by, the gas is released into the atmosphere from the opening of the tip of the discharge tube 55 as shown by the arrow.

The breeding tank 3, the gas treatment unit 41, the series header 25, the header 33, and the pipes 23 and 3 connecting them.
1, 39, etc. constitute one series, and as shown in FIG. 1, in the measuring apparatus 1, three series having the same specifications are arranged. In each series, air is supplied under the same conditions. Of the 3 series, 1 series is used as the experimental series (1), treated mice (black mice) are bred in the breeding tank 3, and 1 series is used as the control experimental series (2) in the breeding tank 3. The untreated mouse (gray mouse) is bred, and the remaining one series is for measuring the total amount of carbon dioxide contained in the gas supplied to the breeding tank 3, that is, a standard series (3 for checking the safety of the device). ), And animals are not bred in the breeding tank 3.

In the experimental series (1) and the control experimental series (2),
Cc in the above formula (1) of carbon dioxide generation is measured,
In the standard series (3), the above formula (1) of carbon dioxide generation
The inside Cb will be measured. By providing the reference series (3), the safety of the device is checked, and the experimental accuracy is remarkably improved as compared with the conventional device.

Reference numeral 57 indicates a pipe, which is a pipe 57.
One end of is connected to the header 33. The other end constitutes an air supply port. A filter 59 and an air pump 61 are provided in the middle of the pipe 57. A carbon dioxide (CO ₂ ) absorbent is attached to the filter 59, and the concentration of carbon dioxide contained in the air supplied to the breeding tank 3 is 0% or less depending on the presence or absence of the absorbent. It can be lowered close. The filter 59 constitutes a carbon dioxide concentration adjusting means.
When the air pump 61 is operated, air is accumulated in the header 33. Pipe 57, air pump 61, header 3
3, the pipe 31, and the valve 35 constitute gas supply means to the breeding tank 3.

The procedure of the measuring work using the measuring apparatus 1 will be described below. First, the weight of the collection cylinder 49 (filled with soda lime (NaOH)) and the water absorption cylinder 51 of each series is measured, and then each series is arranged so as to have the configuration shown in FIG. The weight is measured with an electronic balance having a sensitivity of 10 mg or less. After the treated mouse and the untreated mouse are placed in the cages of the series (1) and (2), respectively, the cage 17 is placed in the container body 5 and the lid 7 is closed. Further, the cage of the series (3) is put in the container main body 5 while being empty, and the lid 7 is closed. Then, the inside of the breeding tank 3 is controlled to a constant temperature by the heater 19 and the temperature sensor 21.

After the above preparatory work is completed, when the air pump 61 is operated, the air whose carbon dioxide (CO ₂ ) concentration is adjusted by the filter 59 is sent to the header 33 through the pipe 57. The air accumulated in the header 33 is adjusted in amount by the valve 35 of each series (1), (2), and (3), and then the series header 2 is passed through the pipe 31.
5, the test liquid L is contained therein, then passes through the pipe 23, is sterilized by the filter 29, and finally supplied into the breeding tank 3.

The gas (air) discharged from the breeding tank 3 through the pipe 39 first flows into the ammonia absorption bottle 43 of the gas processing section 41, and ammonia (NH ₃ ) is removed. Further, after water (H ₂ O) is removed through the first dehumidifying cylinder 45 and the second dehumidifying cylinder 47, the water reaches the collecting cylinder 49. In the collecting cylinder 49, carbon dioxide (CO ₂ ) causes a chemical reaction, and N
It is collected as a ₂ CO ₃ . Further, after the moisture generated by the chemical reaction is absorbed by the moisture absorbing cylinder 51, it is discharged as exhaust gas from the front end opening portion of the discharge cylinder 55 into the atmosphere as shown by the arrow.

After the end of the predetermined measurement time, the collection cylinder 49 and the water absorption cylinder 51 of each series are removed, and the weight is measured in the same manner as before the measurement work. Then, in consideration of the conversion to sodium carbonate (Na ₂ CO ₃ ) from the weight change data before and after the measurement operation of the collection cylinder 49 and the water absorption cylinder 51 of each series, the collection of each series The amount (weight) of carbon dioxide (CO ₂ ) trapped in the cylinder 49 is calculated. The amount of carbon dioxide obtained by subtracting the amount of carbon dioxide generated from the series (3) from the amount of carbon dioxide generated from the series (1) indicates the amount of carbon dioxide generated by respiration during the breeding period of the minimal animal for treatment experiment, and the series (2)
The amount of carbon dioxide obtained by subtracting the amount of carbon dioxide generated in the series (3) from the amount of carbon dioxide generated in (3) indicates the amount of carbon dioxide generated by respiration during the breeding period of the untreated minimal animal. The carbon dioxide amount obtained by subtracting the latter from the former indicates the treatment effect. By performing this appropriately, continuous measurement becomes possible. As a result, the gravimetric method quantifies the respiratory carbon dioxide emissions of laboratory animals.

Further, the valve 13 is opened, and urine and feces of the experimental animal are collected from the collection port 11, and this is also quantified. Therefore, according to the measuring device 1, not only the amount of carbon dioxide generated by respiration but also the metabolites such as urine and feces can be quantitatively analyzed.

Although the embodiment of the present invention has been described in detail above, the specific configuration is not limited to this embodiment, and there are design changes and the like without departing from the scope of the present invention. Also included in the present invention. For example, although three sequences are configured as one set in the above embodiment, the number of sets may be increased. If the number of sets is increased, the measurement work under several kinds of experimental conditions can be carried out concurrently, and the measurement work can be made efficient. Further, it is possible to eliminate uncertain factors due to the measurement time. Further, by making a work in the breeding tank, electric shock stress, heat stress, etc. can be given to the experimental animal.

[0034]

According to the measuring apparatus of the present invention, as compared with the apparatus for measuring radioactive carbon dioxide, the measuring apparatus is small and has excellent convenience, maintenance is easy, and the apparatus price can be set low. Further, according to the measuring device of the present invention, since carbon dioxide is captured by a chemical reaction and quantified by the gravimetric method, even if a controller for air supply that is currently on the market is used, a very small animal can be detected. For example, it can be accurately measured from one mouse. Specifically, 10 mg / day to 10 g /
It is possible to accurately measure the carbon dioxide generation amount of about day. Further, according to the measuring apparatus of the present invention, since carbon dioxide is collected by a chemical reaction and quantified by the gravimetric method, extremely stable measurement work can be performed for a long time. In addition, the continuity of the measured data will not be lost even if an accident that does not affect the life of the laboratory animal occurs.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a measuring device according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing one series in the apparatus of FIG.

[Explanation of symbols]

1 Measuring Device for Carbon Dioxide Generation by Breathing of Experimental Animal 3 Breeding Tank 5 Container Body 7 Lid 9 Pipe 11 Urine and Feces Collection Port 13 Valve 15 Support 17 Cage 19 Heater 21 Temperature Sensor 23 Pipe 25 Series Header 27 Gas Analysis port 29 Aseptic filter 31 Pipe 33 Header 35 Valve 37 Cumulative flow meter 39 Pipe 40 Gas analysis port 41 Gas treatment part 43 Ammonia absorption bottle 45 First dehumidification cylinder 47 Second dehumidification cylinder 49 Collection cylinder 51 Moisture absorption cylinder 53 Connection part 55 Discharge cylinder 57 Pipe 59 Filter 61 Air pump

   ─────────────────────────────────────────────────── ─── Continued front page    (71) Applicant 598176178             Yasuhiro Tsukamoto             1-20 Tamachi, Shizuoka City, Shizuoka Prefecture (71) Applicant 501423894             Atsushi Murakami             12-5 Shimizu Umedacho, Shizuoka City, Shizuoka Prefecture (72) Inventor Shogo Uematsu             1-519 Orido, Shimizu City, Shizuoka Prefecture (72) Inventor Atsushi Murakami             12-5 Umedacho, Shimizu City, Shizuoka Prefecture F-term (reference) 2B101 AA11 FA01 FA07                 2G042 AA01 BB05 CB01 FA02 HA02                 2G045 AA29 CB23 DB01 GC07 HA14

Claims (8)

[Claims] 1. A hermetically sealed breeding tank, a gas supply means for supplying an oxygen-containing gas to the breeding tank at a constant air supply amount, and carbon dioxide contained in the exhaust gas from the breeding tank by a chemical reaction. And a series having a collecting means for converting into a form that can be quantified by the gravimetric method and then collecting the same, and based on the weight measurement of the carbon dioxide collected by the collecting means, the carbon dioxide by breathing of an experimental animal is provided. A device for measuring the amount of carbon dioxide produced by respiration of an experimental animal, which is characterized by calculating the amount of carbon produced. 2. The apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to claim 1, wherein an experimental series in which treated animals for experiment are bred in a breeding tank and a control experiment in the breeding tank are used. 2. A measuring device comprising three series of a control series for raising untreated animals and a reference series for measuring the total amount of carbon dioxide contained in the oxygen-containing gas supplied to the breeding tank. 3. The apparatus for measuring the amount of carbon dioxide produced by respiration of a laboratory animal according to claim 1 or 2, further comprising carbon dioxide for controlling the concentration of carbon dioxide contained in the oxygen-containing gas supplied to the breeding tank. A measuring device comprising a carbon concentration control means. 4. The apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to any one of claims 1 to 3, further comprising a sterilization processing means for sterilizing the gas supplied to the breeding tank. A measuring device comprising. 5. The apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to any one of claims 1 to 4, wherein the breeding tank has a two-layer structure of an outer closed container and an inner cage. The cage is detachably mounted inside the closed container. 6. The apparatus for measuring the amount of carbon dioxide produced by respiration of a laboratory animal according to claim 1, wherein the breeding tank is made of a heat-resistant material. 7. The apparatus for measuring the amount of carbon dioxide produced by respiration of an experimental animal according to any one of claims 1 to 6, wherein a means for collecting urine and feces of the experimental animal is provided below the breeding tank. Measuring device characterized in that 8. A measuring device for measuring the amount of carbon dioxide generated by respiration of an experimental animal according to any one of claims 1 to 7, which is for a very small animal.