Method for manufacturing edema model rats

Abstract

Edema reduces quality of life. To alleviate this, the development of drugs such as edema preventatives or anti-edema medications is necessary, but one of the obstacles to their development has been the lack of animal models. [Solution] The process of anesthetizing the rat, A rat model of edema can be obtained by a method for manufacturing edema model rats that includes the step of fixing the rat and immersing the part of the rat from the calcaneus or from the carpal bones in cold water for 15 minutes or more.

Description

[Technical Field]

[0001] This invention provides a method for manufacturing an edema evaluation model animal necessary for resolving edema, and specifically a method for intentionally inducing edema in rats. [Background technology]

[0002] Swelling (or edema) can manifest as a symptom of various diseases. Therefore, treating the underlying disease causing the swelling should take priority over treating the swelling itself. Patent Document 1 describes creating a rheumatoid arthritis model mouse by injecting bovine type II collagen into mice.

[0003] Furthermore, Patent Document 2 describes how edema induced by an antigen-antibody reaction is expressed in mice using egg albumin. These methods express edema models so that edema can be used as an evaluation indicator of the degree of treatment. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2016-526026 [Patent Document 2] Special Publication No. 2003-524653 [Overview of the project] [Problems that the invention aims to solve]

[0005] Edema can occur even without being a symptom of a serious illness. For example, it can occur when doing so-called "water work" involving cold water, or when working with bare hands in cold weather. In such cases, edema occurs, albeit temporarily, and quality of life decreases. To alleviate this, the development of drugs such as edema preventatives or anti-edema medications is necessary, but one of the obstacles to their development is the lack of animal models. [Means for solving the problem]

[0006] This invention was conceived in view of the above problems and provides a method for producing an animal model in which edema caused by cold water or edema caused by prolonged immobilization is induced. The rat in which edema has been induced is called an "edema model rat."

[0007] More specifically, the method for producing an edema model rat according to the present invention is as follows: The process of anesthetizing rats, The method is characterized by the step of fixing the rat and immersing the part of the rat from the calcaneus or from the carpal bones in cold water for 15 minutes or more. [Effects of the Invention]

[0008] This invention allows for the production of rats as a model animal for cold-induced edema, which can contribute to the development of drugs for this condition. [Brief explanation of the drawing]

[0009] [Figure 1] This is a photograph showing a method for inducing edema in rats using cold water. [Figure 2] This photograph shows a method for inducing edema in rats through immobilization. [Figure 3] This is a photograph showing an example of swelling that occurred. [Figure 4] This is a time chart showing a schedule for soaking your feet in 15°C cold water for 15 minutes. [Figure 5] This graph shows the percentage increase in the thickness of the instep after immersing the feet in 15°C cold water for 15 minutes. [Figure 6] This table shows the incidence rate of edema. [Figure 7] Figure 6 is a graph of the same data. [Figure 8] This is a time schedule for soaking your feet in 10°C cold water for 15 minutes. [Figure 9] This graph shows the percentage increase in the thickness of the instep after immersing the feet in 10°C cold water for 15 minutes. [Figure 10] It is a graph showing the increase rate of the nail thickness when the feet are immersed in 10°C cold water for 15 minutes. [Figure 11] It is a table showing the occurrence rate of swelling. [Figure 12] It is Figure 11 in the form of a graph. [Figure 13] It is a diagram showing the time schedule when the feet of rats without ovariectomy are immersed in 10°C cold water for 15 minutes. [Figure 14] It is a table showing the occurrence rate of swelling. [Figure 15] It is the swelling occurrence rate in Figure 14 in the form of a graph. [Figure 16] It shows the time schedule when swelling is induced by plaster cast. [Figure 17] It is a table showing the results when induced by plaster cast. [Figure 18] It is a diagram showing the time schedule of a test for shortening the plaster cast test time. [Figure 19] It is a graph showing the swelling of the feet after 24 hours and 48 hours. [Figure 20] It is a diagram showing the time schedule of a test for further shortening the plaster cast test time. [Figure 21] It is a graph showing the swelling of the feet 1 hour after plaster cast treatment.

Embodiments for Carrying Out the Invention

[0010] The method for producing a rat model of edema according to the present invention will be described below with reference to drawings and examples. The following description illustrates one embodiment and one example, and the present invention is not limited to the following description. The following description may be modified without departing from the spirit of the invention. Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included within the technical scope of the present invention. All references cited herein are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," it means "A or greater and B or less."

[0011] The method for producing the edema model rat according to the present invention is carried out by inducing edema in rats. Rats have the advantages of being easier to work with than mice because their limbs are larger, and they are relatively inexpensive to obtain. Above all, it has been confirmed that edema can be induced in rats using a relatively simple method with cold water.

[0012] Edema in rats is typically induced in the hind legs, beyond the tarsal bones (towards the claws). It can also be induced in the forelegs, beyond the carpal bones (towards the claws). This is because these areas lack fur, the skin is exposed, and it is easier to determine whether or not edema has occurred.

[0013] Figure 1 shows a method for inducing edema in rats using cold water. Figure 1(a) shows the treatment table used. The legs 10 and wire mesh 12 are fixed and placed in a water tank, after which water at a constant temperature is added to the tank. A beaker is placed between the legs 10 and filled with water at a constant temperature. The rat to be treated is anesthetized and then placed on the wire mesh 12. Note that the anesthesia process may involve administering a sedative. Figure 1(b) is a photograph of the rat being treated. The rat's hind legs are hung downwards through the holes in the wire mesh 12 so that the part below the calcaneus is submerged in water. This submerged part is called the "immersion part".

[0014] Figure 2 shows a method for inducing edema in rats through prolonged immobilization. This method involves straightening the bends in the tarsal bones of the hind legs and the bends in the tibia and femur, then applying splints and fixing them with a cast. Figure 2(b) shows the cast covered to prevent gnawing. By keeping the rats in this state for a certain period, edema caused by immobilization can be induced.

[0015] Figure 3 shows the condition of the hind legs of rats that have been induced with edema. Compared to before treatment (normal), the toes and top of the foot are enlarged after treatment (edema). [Examples]

[0016] Next, we will explain the occurrence of edema in rats with the help of examples. <Creation of model rat C for swelling caused by cold> The edema model rats were treated using the treatment table shown in Figure 1, following the procedure below. (C-1) Rats were administered a three-component mixture of anesthetics. (C-2) The fixed legs 10 and wire mesh 12 were placed in the water tank. (C-3) Water and ice were placed in the tank, and the water temperature was adjusted to the specified temperature (10°C or 15°C). (C-4) A paper towel was placed on top of the wire mesh 12. (C-5) The rats were placed on paper towels, their feet were suspended through the holes in the wire mesh 12, and they were immersed in the water tank. (C-6) We waited there for 15 minutes. (C-7) Remove the rat from the paper towel (wire mesh 12) and wipe off any water droplets without applying pressure to the immersion area.

[0017] We also prepared female rats from which the ovaries had been removed. This was done because there is some data (though not statistically confirmed) suggesting that swelling due to cold and lack of exercise is relatively common in postmenopausal women, and we prepared this model to examine the validity of this claim.

[0018] Figure 4 shows the timeline for preparing the model rats and the schedule for the cold water test. Ovariectomy was performed by dividing the rats into two groups: one group (called "OVX1") had both ovaries removed simultaneously after one week of acclimatization upon arrival, and the other group (called "OVX2") had one ovary removed at that time, followed by the removal of the other ovary one week later. A control group (called "Sham") was also prepared, in which only laparotomy was performed one week after acclimatization, and no ovariectomy was carried out. Each group consisted of n=7-8 rats.

[0019] Cold water testing was conducted once a week. However, the 5th and 8th weeks from the date of arrival were designated as rest weeks, and cold water testing was not performed during these weeks.

[0020] Next, we will explain the evaluation of edema. Edema can be evaluated by the difference in foot thickness before and after treatment. On the other hand, since the evaluation model involves experiments with multiple animals, an evaluation method that is less affected by individual differences is also necessary. Furthermore, if we try to evaluate edema by the difference in foot thickness, we will have to actually measure the thickness of the foot. Therefore, an evaluation method that is less affected by measurement errors is necessary. For the measurement of foot thickness, we decided on a measurement point and measured the thickness of the foot at that point using calipers.

[0021] To minimize the impact of measurement errors, we first determined the rate of increase in edema (hereinafter also simply referred to as "rate of increase") and a reference value (hereinafter also simply referred to as "reference value") for evaluating a group of multiple animals. For individual A, the thickness of the foot before treatment was denoted as L, and the thickness of the foot after treatment was denoted as M. Measurements were taken five times consecutively at the same location. The rate of increase Ri is expressed by the following equation (1).

[0022]

number

[0023] L k This is the k-th measurement before treatment, and M k This is the k-th measurement after the procedure.

[0024] Also, the reference value ST was obtained as follows. Assume there are N individuals. The k-th error LE of the n-th individual before treatment n k was obtained as shown in the following equation (2). Also, the k-th error ME of the n-th individual after treatment n k was obtained as shown in the following equation (3).

[0025]

Equation

[0026] Next, the largest value among LE n k was defined as LE n . Similarly, the largest value among ME n k was defined as ME n .

[0027]

Equation

[0033] <15℃-15min test> Figures 5(a), 5(b), and 5(c) show graphs representing the results for weeks 6, 7, and 9, respectively, when the cold water temperature was set to 15°C (foot immersion time was 15 minutes). In all figures, the horizontal axis represents the group type (Sham, OVX1, OVX2), and the vertical axis represents the rate of increase (%). Throughout the data, "*" indicates P<0.05.

[0034] Each group has four measurement results, which, from left to right, are: before the cold water test (marked "pre"), immediately after the cold water test (marked "0"), 120 minutes after the cold water test (marked "120"), and 240 minutes after the cold water test (marked "240"). All values ​​are calculated with the value before the cold water test set to 100%.

[0035] Figure 6 shows a table comparing these results to the weekly reference value STweek. In the table, the reference increase rate is the reference value ST. The overall edema rate is the percentage of rats in the week in which the experiment was conducted that were judged to have edema at 0 min based on the reference value ST.

[0036] In the Sham group, the incidence of edema was 16.7% at week 6, rising to 80.0% at week 7. However, this decreased to 57.1% at week 9. In the OVX1 group, the incidence was 16.7% at week 6, rising to 100% at week 7, and remaining at 66.7% at week 9. In the OVX2 group, the incidence was 50.0% at week 6, rising to 80.0% at week 7, and decreasing to 60.0% at week 9.

[0037] Figure 7 shows this data graphically. The horizontal axis represents the week of the cold water test, and the vertical axis represents the edema incidence rate (%). The edema incidence rate is the percentage of cases where the value exceeded the reference value ST in the table in Figure 6. Four values ​​are shown for each week, from left to right: Sham, OVX1, OVX2, and ALL. ALL represents the edema incidence rate for all groups combined for that week.

[0038] This study showed that 15 minutes of cold water at 15°C could induce edema with a high probability by the 7th week after admission. Furthermore, the group in which both ovaries were removed at once (OVX1 group) was able to induce edema even more reliably by the 7th week. This edema almost completely subsided after 24 hours.

[0039] <10℃-15min test> Figure 8 shows the schedule for the test groups when the water temperature was set to 10°C. In the 10°C test, OVX1 was not included, and only the OVX2 group and the Sham group were used. There was a rest period for the third week after acceptance, and the test was conducted continuously from the fourth week to the ninth week.

[0040] Figures 9 and 10 show the results of the growth rate. Figures 9(a) and 9(b) show the results for weeks 5 and 6 after acceptance, respectively, and Figures 10(a) and 10(b) show the results for weeks 7 and 8 after acceptance, respectively. Throughout Figures 9 and 10, the horizontal axis represents the group and the vertical axis represents the growth rate (%). Also, throughout, "*" represents P < 0.05 and "△" represents P < 0.1.

[0041] In Figures 9 and 10, the horizontal axis has four values, from left to right: the results before the cold water test (labeled "pre"), immediately after the cold water test (labeled "0"), 90 minutes after the cold water test (labeled "90"), and 180 minutes after the cold water test (labeled "180").

[0042] Figure 11 shows a table comparing the results with the weekly baseline value STweek. Figure 12 shows a graph of this data. In Figure 12, the horizontal axis represents the measurements for each week, and the vertical axis represents the edema incidence rate (%). From left to right, each week represents the Sham group, the OVX2 group, and the ALL group. From these results, it can be seen that at 10℃, the edema incidence rate was highest in week 6.

[0043] <In cases where the ovaries are not removed> Creating edema model rats requires the cumbersome process of removing the ovaries each time. Therefore, we investigated the case where the ovaries were not removed. The procedure for creating edema model rats followed (C-1) to (C-7). However, the water temperature was only 10°C. In addition, the cold water test was performed on both feet of each rat. As a result, the number of samples was greater than the number of rats. Figure 13 shows the timeline of the preparation of the model group and the schedule of the cold water test.

[0044] Seven 6-week-old female rats were used. As shown in Figure 13, the rats underwent a one-week acclimatization period after arrival. The cold water test was then conducted from the first week after arrival. The cold water test was performed weekly until the sixth week after arrival.

[0045] Figure 14 shows a table representing the results, and Figure 15 shows a graph representing the incidence of edema. Referring to Figure 15, the horizontal axis represents the week of the cold water test, and the vertical axis represents the incidence of edema (%). Edema was induced with a probability of over 80% from the first week. Furthermore, an incidence of edema was recorded at 100% in the third week. From the fourth week onward, the incidence of edema gradually decreased.

[0046] These results show that edema was induced at an earlier stage than in OVX1 (a model rat from which two ovaries were removed simultaneously) in the 15°C-15 minute cold water test shown in Figure 7. Furthermore, edema was induced with a higher probability than in the 10°C-15 minute cold water test shown in Figure 12.

[0047] <Creation of a edema model rat F by fixation> The immobilized edema model rat F was prepared using the following procedure. (F-1) Rats were administered a three-component mixed anesthetic. (F-2) A wooden dowel was loosely secured to the heel (tarsal bone) of the left foot with an adhesive bandage, leaving the toes exposed. (F-3) The knee (femur and tibia) was fixed in the same way. (F-4) Wrap the elastic bandage in multiple layers starting from the toes, smoothing out any unevenness in the wooden dowel to make it thicker. (F-5) I moistened the plaster cast with 37℃ water, wrung it out firmly, and wrapped it around the foot starting from the toes, rubbing it with my fingers to help it adhere. (F-6) Leave it to dry for 15 minutes, then wrap wire mesh around it and tighten it with wire. Tighten the base of the legs firmly to prevent them from coming off. (F-7) The analgesic atipamezole (Antisedan®) was administered to rats. (F-8) The patient was observed daily after the procedure, and if the fixation came loose, the procedure was reapplied each time.

[0048] Figure 16 shows the schedule for the gypsum test. Four-week-old male SD rats were used for the gypsum treatment. After acceptance and acclimatization for 6 days, they were switched to a high-fat diet and fed for 1 week. In the second week, they were divided into a group that received gypsum treatment and a group that did not. Subsequently, in the third and fourth weeks, the gypsum was removed and visual inspection was performed. The results are shown in Figure 17. "Normal" represents the group without gypsum, and "Gypsum" represents the group that received gypsum treatment. Edema occurred in 100% of the rats after 1 week in the group that received gypsum treatment.

[0049] <Consideration of shortening the time 1> As shown in Figure 17, edema due to cast immobilization could be induced within one week of cast application. Therefore, we attempted to induce edema in a shorter time. The manufacturing method for the edema model rats followed the procedures for (F-1) to (F-8). The evaluation items included measuring the thickness and width of the foot with calipers.

[0050] Figure 18 shows the schedule for the cast test (shortened duration). Four 5-week-old male SD rats were used. The first 5 days after arrival were used for acclimatization. A high-fat diet was not given. Cast treatment was performed on the 6th day, and measurements were taken 24 hours later on the 7th day and 48 hours later on the 8th day.

[0051] The size of the feet was measured during the casting process. This data is known as "cast foot pre". After 24 hours, the casts were removed from the four animals and their foot sizes were measured again. This value is known as "cast foot 24h". Subsequently, the animals were cast again, and after 48 hours, the casts were removed from these four animals and their foot sizes were measured again. This value is known as "cast foot 48h". The results are shown in Figure 19.

[0052] Referring to Figure 19, Figure 19(a) shows the measurement after 24 hours, and Figure 19(b) shows the measurement after 48 hours. In both graphs, the horizontal axis represents the sample, "cast foot pre." is the measurement of the foot before casting, and "cast foot 24h" and "cast foot 48h" are the measurements after 24 and 48 hours, respectively. The vertical axis is the measured value (mm).

[0053] In Figure 19, although the expression is not a rate of increase, and therefore the occurrence of edema cannot be directly observed, both the casted foot at 24 hours and the casted foot at 48 hours showed a significant increase in foot thickness compared to before casting, and edema was induced with 100% certainty.

[0054] <Consideration of shortening the time 2> While plaster cast immobilization could induce edema in a short time, we investigated how much shorter this timeframe could be. The manufacturing method for the edema model rats followed the procedures described in (F-1) to (F-8). The evaluation items included measuring the thickness and width of the foot using calipers.

[0055] The schedule for the cast test (shortened duration) is shown in Figure 20. Five-week-old male SD rats were used. The first seven days after arrival were used as an acclimatization period. A high-fat diet was not administered. Casting was performed on the eighth day, and measurements were taken one hour after cast application. The results are shown in Figure 21. Similar to Figure 19, the casted foot was significantly larger than the uncasted control foot one hour later, indicating that edema was successfully induced. [Industrial applicability]

[0056] The method for producing edema model rats according to the present invention can produce rats with edema and can be suitably used in the development of drugs such as edema preventatives.

Claims

1. The process of anesthetizing rats, A method for producing an edema model rat, comprising the step of fixing the rat and immersing the part of the rat from the calcaneus or from the carpal bones in cold water for 15 minutes or more.

2. The method for producing an edema model rat according to claim 1, wherein the rat is female and has had its ovaries removed in advance.

3. The method for producing an edema model rat according to claim 2, wherein the rat is an individual from which two ovaries have been simultaneously removed.

4. The method for producing an edema model rat according to claim 2, wherein the rat is an individual from which one ovary has been removed at one time every week.

5. The process of anesthetizing rats, A method for manufacturing an edema model rat, comprising the step of immobilizing the hind leg or foreleg of the rat with a cast.

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