KR101370696B1 - A method for constructing tinnitus animal models and a method of behavioral test of tinnitus in the same animal models - Google Patents

Abstract

The present invention provides a method of producing a tinnitus animal model using a skinner box and a method of behavioral evaluation of tinnitus using a tinnitus animal model. According to the method of the present invention, by discriminating the sound in the skinner box without electric stimulation for the experimental animals, it is possible to evaluate the behavioral response of the tinnitus animal model without stressing the animals, which is a mechanism research or treatment related to tinnitus Useful for development research.

Description

A method for constructing tinnitus animal models and a method of behavioral test of tinnitus in the same animal models}

The present invention relates to a method of manufacturing a tinnitus animal model using a skinner box and a method of behavioral evaluation of tinnitus using a tinnitus animal model.

Tinnitus is often referred to as "earing" (perception of sound in the absence of an external source of sound signals). Tinnitus (tinnitus), tinnitus, or tinnitus is the recognition of sound in the human ear without a corresponding external sound. To put it plainly, sounds that are not external are heard internally (Jastreboff et al., J Am Acad Audiol ,. 11 (3), pp162-177, 2000). These tinnitus may appear for a moment, disappear, persist, and continue to sound. The height of the sound varies and is usually present in one ear, or can be heard in both ears.

Statistically, about 15-20% of adults experience a variety of tinnitus, 4% of whom have severe tinnitus. In addition, that 70-80% of the cases of hearing loss experienced tinnitus (Demeester K et al., B -ENT., L7, 37-49, 2007). Tinnitus can be divided into subjective tinnitus (or objective tinnitus) that can be heard from the outside and subjective tinnitus (or subjective tinnitus) that can only be heard by the tinnitus patient. In addition, tinnitus can be classified into peripheral tinnitus and central tinnitus based on differences in the way they are perceived by a diseased individual. Peripheral (or cochlear) tinnitus is believed to be derived from the peripheral nervous system and cochlea, and central tinnitus is believed to be derived from the auditory cortex.

An alternative tinnitus that allows the doctor to hear the actual sound from the patient's ear can cause the muscles around the middle ear to 'click' or 'muddy' with convulsions. Some people listen to the sound with a pulse. Pulsatile tinnitus can be caused by changes in blood flow or flow of veins around the ear (Chandler JR, Laryngoscope, 93, pp892-895, 1983), but it can be an acute symptom caused by increased interest in the blood flow in the ear. To date, pathological physiological characteristics of subjective tinnitus are insufficiently understood and the exact pathogenesis is unknown.

Tinnitus is usually a subjective phenomenon, and it is difficult to measure with a general evaluation of the hearing aid. This was regarded as a limiting factor in animal experiments on tinnitus and failed to succeed in the development of tinnitus therapeutics. In clinical practice, the evaluation of tinnitus is assessed by scoring the patient's subjective pain. The condition is often judged clinically as a simple measurement rating from 'mild' to 'extreme' according to realistic difficulties such as sleeping, meditation, and disruption of daily activities. Persistent tinnitus can lead to hypersensitivity, fatigue, and sometimes clinical depression, and hearing may also be heard (McCombe et al., Clin Otolaryngol., 26 (5), pp 388-393, 2001 .; Davis et al. al., Epidemiology of Tinnitus.In: Tyler R, editor.Tinnitus Handbook.San Diego: Singular Publishing Group; 2000. pp1-23).

An animal model of tinnitus was first introduced in the 1990s, suggesting that tinnitus causes tinnitus three hours after administration of a single high-dose (typically 350 mg / kg dose) of aspirin, the main ingredient of sodium salicylate. Reported (Chen, K et al., Hearing Res., 82, pp158-178, 1995). The salicylate model has been used for many studies as acute or short-lived tinnitus due to spontaneous disappearance of tinnitus 24 hours after administration. In the 2000s, chronic tinnitus animal models with excessive noise exposure were introduced (Heffner et al., Hearing Res., 170, pp83-95, 2002; Seki et al., Hear Res., 180, pp28-38, 2003). ). The tinnitus animal model by noise exposure depends on the time or intensity of noise exposure and introduces various protocols for each laboratory studied.

In clinical practice, tinnitus does not have an objective assessment tool, and the tinnitus is evaluated based on the subjective assessment method that the patient complains about. To identify tinnitus as a subjective evaluation method in a tinnitus animal model is to train the animal to respond to sounds using a skinner box and then to assess the behavioral changes in the animal that caused tinnitus. In general, animals have to be used with a cognitive knowledge that can be trained to discriminate sound, and rats have never been used as rats. The skinner box has a structure in which the animal presses the lever and the food is released. The animal is trained to eat the food by pressing the lever only when the sound used for the indication sound is heard. If you press the lever when there is no beeping sound, the electric wires underneath the skinner's box will be energized, causing an electric shock for a short time, which will cause the animal to recognize the wrong behavior. Such training usually takes more than a month and trained animals are generally used in experiments when the success rate reaches 80%. Lukas et al., Hearing Research, 180, pp39-50, 2003; carol et al., Otolaryngol Head Neck Surg., 121, pp457-462, 1999). In addition to the above method, there is an evaluation method using active avoidance. Unlike the skinner box, the evasion reaction training and inspection box has two rooms, and a small life span is connected between the rooms so that animals can move to the opposite room if necessary. It is a structure. When you hear the sound used as audible tone, electric current is applied to the electric line under the sole of the foot to give electric shock for a while, and the animal escapes the electric shock to the opposite room. Trained animals move to the opposite room to hear the sound and not receive electric shock (Guitton et al., J Neurosci., 23, pp3944-3952, 2003; Kizawa et al., Neuroscience, 165, pp1323-1332, 2010 ). To date, there have been only two methods of evaluating behavioral responses in tinnitus animal models, and rats have been reported mainly in the animals used.

Most tinnitus patients belong to subjective tinnitus, and the treatment for subjective tinnitus has been focused on relaxation of tinnitus through rehabilitation. For example, a hearing aid-like device allows the user to hear sounds similar to the tinnitus of the patient at a louder volume than tinnitus, so that tinnitus is not heard. It is also known as tinnitus retraining therapy that treats tinnitus without hearing loss. To date, no tinnitus medication has been approved by the US Food and Drug Administration (FDA). Therefore, research on pharmaceutical products effective for the treatment or prevention of tinnitus is necessary, and related studies should be preceded by the development of an accurate and stable evaluation method of an appropriate tinnitus model and animal model.

In addition, regulations on animal testing have been gradually strengthened internationally in Europe and elsewhere. Also, with the revision of the animal protection law in Korea, regulations on indiscriminate animal testing have been strengthened, and interest in animal test ethics has increased. In particular, there was a request for experimental models to avoid or minimize pain in experimental animals.

Accordingly, under the background of the development of a method for evaluating the behavioral response of a tinnitus animal without electrical stress, the present inventors have developed a behavioral reaction test method that can be evaluated by inducing tinnitus in an experimental animal without electrical stress, thereby validating the present invention. Was completed.

The present invention aims to develop a method for evaluating the behavioral response of a tinnitus animal model without stress on an animal by completing a discrimination training for a sound in a skinner box without electric stimulation using a rat.

The present invention provides a method for producing a tinnitus animal model using a skinner box.

The present invention provides a method of making a tinnitus animal model using a rat without stress on the animal.

The present invention also provides a method for evaluating tinnitus behavior using a tinnitus animal model.

The present invention provides a method of behavioral evaluation of tinnitus without stress on animals using rats.

As used herein, the term “Tinnitus” includes both tinnitus and subjective tinnitus and includes peripheral and central tinnitus. Preferably, it includes subjective tinnitus caused by various causes such as noise, drugs, aging, trauma, viruses and the like.

The present invention is to develop a method for evaluating the behavioral response of a tinnitus animal model without stress to the animal by completing the discrimination training for the sound in the skinner box without electric stimulation using a rat.

The tinnitus animal model of the present invention is made by a method comprising the following steps using a skinner box:

(1) a purifying step of selecting and purifying a test animal having a normal hearing by the electrophysiological hearing test method to the examiner;

(2) when the experimental animal subjected to the purifying step presses the lever in the skinner box to provide food and cue-tone at the same time, wherein the individual who has acquired food for 80 times or more for 15 minutes is selected for the next step, A primary screening step comprising dropping off an individual who has not learned more than 80 times within 7 days;

(3) to the experimental animals subjected to the primary screening step, provide a cue tone for 15 seconds per minute, where the lever in the skinner box is pressed within 1 second while the cue is heard or after the cue is finished (according to: true). Pressing the lever when feeding but without a cueton (false response) selects for the next step an object that does not provide food and that the wrong response is 20% or less of the total number of lever presses during the 15-minute training period. And a second sorting step comprising dropping off the individual with a wrong response within 7 days more than 20% of the total number of lever presses; And

(4) Irregularly provide three times the cue tone three times per minute to the experimental animal that has undergone the second screening step, where the lever in the skinner box is pressed within one second while the cue is heard or after the cue is finished. true response) If the lever is pressed when food is provided but there is no cueton (false response), the subject is not provided with food and the wrong response is less than 20% of the total number of lever presses during the 15-minute training period. A final screening step comprising selecting, but dropping, subjects with less than 20% of the total number of lever presses within 7 days.

The purifying step is an electrophysiological hearing test using only animals having a hearing threshold of 30 dB or less for the click stimulus, and purifying by the examiner simultaneously listens to the tester's voice and feeds the tester's voice every day. It is done by stroking and only by putting up to 3 experimental animals in the cage.

The electrophysiologic auditometry is a test for predicting auditory ability based on an electrophysiological potential for electrical stimulation, an impedance telemetry test, an electrical induced spinal muscle reflex test, and an induced auditory brainstem test. And electroinduced complex action potential tests, and these methods are known in the art and are described, for example, in Hong and Kang (Neuroscience Letters 431 (2008) 268-272).

Experimental animals used in the present invention may be used, including rodents, mammals, rats are preferred.

Skinner box used in the present invention is known to those of ordinary skill in the art as a training and evaluation device for behavioral evaluation of animal models, the exemplary structure is shown in FIG. . As can be seen in Figure 1, the skinner box is mainly used as a device for evaluating the cognitive ability of the animal as a behavioral response, the structure of the acrylic box and the acoustics of the sound in the box, the animal's foot button (trigger, In the present specification, the press "Lever") when a small amount of food is dropped one by one, the bottom is provided with an electrical stimulation plate that can give an electrical stimulation if necessary. In order to assess subjective tinnitus, approximately one month's training is performed to allow animals to behave in response to subjective tinnitus.

In the primary screening step, the cue tone is provided for 3 seconds at 60 dBA in 8 kHz pure tone.

In the second sorting step, the cue tone is provided irregularly within 1 minute and is provided as a continuous sound for 15 seconds at 60 dBA with 8 kHz pure tone.

In the final screening step, the cue tone is provided irregularly three times for three seconds for one minute, and is provided for three seconds of continuous sound at 60 dBA in 8 kHz pure tone.

As an aspect of the present invention, it is preferable to provide only the water and fast for 15 to 18 hours before performing the steps (2) to (4).

In addition, as an aspect of the present invention, the steps (2) to (4) is to remove the ambient light in a quiet place of less than 30 dBA, to provide lighting only in the skinner box, to hear a white noise of 40 dBA It is desirable to train at.

The present invention also provides a tinnitus behavioral assessment method characterized by using a tinnitus animal model constructed by the method described above. This is useful for mechanism studies or tinnitus-related studies. For example, it can be used to evaluate behavioral responses to assess drug effects on subjective tinnitus.

The rats used in the present invention were subjected to females at 4 weeks of age, and only rats with normal hearing were used for the evaluation of electrophysiological hearing test before training. Normal hearing should be below 30 dB. Perform 10 minutes of fine training each day for 3 days prior to this training. Purification training aims to get rid of the fear of human beings and make them familiar by stroking one by one.

The main training from step (2) to step (4) is started for 5 week-old females who have been trained. 18 hours of fasting before training and 15 minutes of free food and water to eat. The duration of each phase of training may vary according to individual differences in rats, but animals that do not pass each phase in 10 days are eliminated. After training up to level 3, you can use it for experiments. The experiment usually takes about 7 days and can be retested after 3-4 days of rest. However, if you participate in the experiment more than 3 times, the rat may discriminate between the sound used in the experiment and the tinnitus sound. Therefore, the experiment should be used only 3 times or less. Existing skinner training is trained in parallel with the electrical stimulation, while the present invention can complete the training without electrical stress to the rat and can be used in the experiment.

As regulations on animal experiments are being tightened, the method of making this tinnitus model and the behavioral evaluation method of tinnitus using the tinnitus animal model have completed sound discrimination training in the skinner box without electric stimulation for experimental animals. In addition, the behavioral response of a tinnitus animal model can be evaluated without stressing the animal. This is useful for mechanism studies or tinnitus-related studies.

1 is a diagram showing a skinner box as a training and evaluation device for behavioral evaluation of a tinnitus animal model.
2 is a schematic diagram showing a training procedure for behavioral evaluation of a tinnitus animal model.
Figure 3 is a diagram showing the results of the two-step training for behavioral evaluation of the tinnitus animal model. After more than 80 times of stage 2 training, it entered stage 3.
Figure 4 is a diagram showing the results of the three-step training for behavioral evaluation of tinnitus animal model. The third stage of training resulted in stage 4 when the false positives dropped below 20%.
5 is a view showing the results of the four-step training for behavioral evaluation of the tinnitus animal model. The test was performed when the false positives were lowered to 20% or lower as a result of the four-stage training.
Figure 6 is a graph showing the number of positive and gastric reactions of behavioral responses in the evaluation of trained animals in a tinnitus animal model. (salicylate 350: salicylate 350 mg / kg treated group, no treatment: untreated control).
Figure 7 is a graph showing the positive and gastric response of behavioral responses in the evaluation of trained animals in a tinnitus animal model. (salicylate 350: salicylate 350 mg / kg treated group, no treatment: untreated control).
Figure 8 is a graph showing the results of the two-stage training of animals that did not go through the first step of the training procedure for behavioral evaluation of the tinnitus animal model.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, these Examples and Test Examples are for illustrating the present invention, and the scope of the present invention is not limited to these Examples and Test Examples.

[ Example ]

Example  1. Training procedure for behavioral evaluation of tinnitus animal model

Rats used for training used a five-week-old cognitive development, fasted for a period of time, and trained in a skinner box for 15 minutes each day. The training stage consisted of four stages. In this training, the training was conducted by purely acquiring food without using electrical stimulation to reduce animal stress.

As confirmed in FIG. 2, in step 1, the experimental animal is purified to the experimenter and external sounds, and in step 2, the lever is pressed in the skinner box to obtain food, and when the food is obtained, a constant sound is simultaneously provided. We trained to recognize cue tones together. In stage 2, if food was acquired more than 80 times in 15 minutes, the process proceeded to stage 3.

In step 3, the same cue tone used in step 1 was played for 1 minute and 15 seconds, and the food was released when the lever was pressed only when there was a cueton. I did not. In step 3, if there was no cue tone, the false response of pressing the lever lowered to 20% or less compared to the total number of lever presses, and then step 4 was entered.

In the fourth stage, the cue tone was played for 1 minute, 3 seconds, 3 times, totaling 9 seconds, and the cue tone gave irregular time intervals. In this case, only when the cuton was played, the lever was pushed to feed the food, and the test was conducted when the rate of the erroneous reaction was lowered to 20% or less. The test repeated four steps for the same week, evaluating true and false positives.

Example  2. Results of the two-stage training for behavioral evaluation of tinnitus animal models

Rats used for training used six 5 week old rats. The electrophysiological hearing test selects individuals whose hearing threshold is 30 dB or less with respect to the click stimulus, and simultaneously listens to the examiner's voice at the time of feeding. Was performed. Only three experimental animals were placed in the cage. Fasting for about 15 to 18 hours and then trained in a skinner box for 15 minutes each day. In the second stage, the lever was pressed in the skinner box to obtain food, and when the food was obtained, a certain sound was provided at the same time to train the sound (cuton) together with the food.

As shown in FIG. 3, as a result of training, five of six rats obtained food more than 80 times in three days, and one obtained more than 80 times in four days. According to the cognitive difference of the rat, there was a slight difference in the performance of the second stage.

Example  3. Results of three levels of training for behavioral evaluation of tinnitus animal models

Six rats that successfully performed step 2 in Example 2 were trained in a skinner box for 15 minutes every day after fasting for a period of time even in step 3. In step 3, if there was no cue tone, the false response of pressing the lever lowered to 20% or less compared to the total number of lever presses, and then step 4 was entered.

As shown in FIG. 4, as a result of training, all 6 mice had a lowered false positive rate of about 10% at the 5th day of the 3rd training and showed a similar tendency at the 6th day. As a result of the same training on the 6th day to confirm the training result on the 5th day, all 6 animals terminated the 3rd stage training.

Example  4. Results of four levels of training for behavioral evaluation of tinnitus animal models

Six rats that successfully performed step 3 in Example 3 were trained in a skinner box for 15 minutes every day after fasting for a period of time even in step 3. In step 4, the cue tone was played three times per minute for three seconds, requiring higher cognitive abilities than step 3. As in step 3, training was terminated when the false response fell below 20% compared to the total number of lever presses.

As shown in FIG. 5, the results of the training showed that the six rats had lowered the erroneous reaction rate to about 10% on the 5th day of the 4th stage of training and showed a similar tendency on the 6th day. As a result of the same training on the 6th day to confirm the training results on the 5th day, all 6 animals were terminated in the 4th stage training.

[ Test Example ]

Test Example  1. The positive and negative responses in the evaluation of behavioral responses after tinnitus induction in a tinnitus animal model Oban Yeah result comparison

The behavioral response evaluation performed the same four steps of tinnitus training for nine days, where the positive reaction refers to the number of times the lever is pushed to acquire food only when there is a cueton, and the misreaction refers to the number of times the lever is pressed when there is no cueton. In tinnitus-induced animals, the positive response to cutone tends to be similar to or slightly reduced before tinnitus is induced, and misreaction of lever-pressing in the absence of cutone tends to increase. At this time, the animal misidentifies the tinnitus sound as a cue tone and shows the act of pressing the lever to confirm the tinnitus induction.

In order to cause tinnitus, fasting animals were divided into two groups, and one group was orally administered only 350 mg / kg of salicylate, which is generally used for tinnitus-inducing animal models, 3 hours before the test. The second group was tested with nothing administered. The first 0 to 1 days of the test period were evaluated using pre-test data, salicylate was administered only for 2 to 5 days, and the tendency to recover without dosing on 6 to 8 days was evaluated.

The measured value on the second day is shown in FIG. 6, and the measured value for the eight days in which the experiment was performed is graphically shown in FIG. 7.

6 and 7, the salicylate-only group significantly lowered the positive response compared to the control group, and the false positives were significantly higher, confirming the tinnitus in the tinnitus animal model. As shown in FIG. 7, the positive response of the group that received salicylate only for 3 days after the end of the dose gradually increased and the false positive gradually decreased so that the tinnitus disappeared completely on the third day.

As described above, when the behavioral reaction test using the skinner box showed a false positive reaction due to tinnitus, it could be used as an index for tinnitus evaluation in a tinnitus animal model, and a false positive due to tinnitus also appeared in a conditional avoidance reaction using a mouse. Mouse models can be used for research on the mechanism of tinnitus or for the development of therapeutics.

[ Comparative Example ]

Comparative Example  1. Not going through the first step On the rat  Stage 2 training results for behavioral assessment

The same experiment as in Example 2 was conducted except that the first step was not performed. The results are shown in Fig. There were no more than 80 rats after training for 5 days, and there were more than 80 rats in the middle, but the behavioral response was not stable.

Claims (7)

Method of making a tinnitus animal model using a skinner box, comprising the following steps:
(1) screening experimental animals with normal hearing by electrophysiological hearing test;
(2) placing the experimental animal with normal hearing screened in the above step in the skinner box, press the lever in the skinner box to provide food and cue-tone at the same time, the individual who learned the food more than 80 times 15 minutes Selecting the first step;
(3) Providing a cue tone for 15 seconds per minute to the experimental animal selected in the primary screening step, and pressing the lever within 1 second while the cue is heard or after the cue is finished (true response). Pressing the lever when there is no cueton (false response) to prevent food from being provided, and selecting the individual whose false response is 20% or less of the total number of lever presses during the 15 minute training time; And
(4) Irregularly provide 3 cues three times per minute to the experimental animal selected in the second screening step, and press the lever within one second while the cue is heard or after the cue is finished (true response). Press the lever when there is no cueton (false response) so that no food is provided, resulting in the selection of a tinnitus animal model for individuals whose erroneous response is less than 20% of the total lever presses during the 15-minute training period. step.  The method of claim 1, wherein the cuetone of the primary screening step is provided for 3 seconds at 60 dBA with 8 kHz pure tone. The method of claim 1, wherein the cuetone of the second screening step is irregularly provided within 1 minute and is provided as a continuous sound for 15 seconds at 60 dBA with 8 kHz pure tone. The method of claim 1, wherein the cue tone of the final screening step is irregularly provided three times for three minutes each for one minute, and is provided for three seconds of continuous sound at 60 dBA to 8 kHz pure tone. How to make. The skinner box according to any one of claims 1 to 4, further comprising the step of providing only the water to the experimental animal and fasting for 15 to 18 hours before performing steps (2) to (4). How to make a tinnitus animal model. The method according to any one of claims 1 to 4, wherein steps (2) to (4) remove ambient light in a quiet place of 30 dBA or less, provide lighting only in a skinner box, and have a white color of 40 dBA. Method of producing a tinnitus animal model using a skinner box, characterized in that the training in the situation of noise. The tinnitus animal model produced by the method of any one of Claims 1-4 is used, The screening method of the tinnitus therapeutic agent.

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