Tree shrew depression model making method

Abstract

The invention discloses a tree shrew depression model modeling method which comprises the following steps: (1) dissolving 20 mg of corticosterone in 10 microliters of DMSO (dimethylsulfoxide), and fixing the volume to 1 ml by normal saline; the method comprises the following steps: dissolving 100 mg of estradiol in 1 ml of DMSO (Dimethylsulfoxide) to prepare 100 mg / ml of estradiol stock solution; and (2) carrying out subcutaneous injection on the corticosterone solution prepared in the step (1) to tree shrews at 14: 00-16: 00 in the afternoon every day at the injection amount of 27 mu g / kg, diluting the estradiol stock solution prepared in the step (1) with normal saline after the corticosterone solution is injected for 1 hour, then carrying out subcutaneous injection on the tree shrews at the injection amount of 50-75 mu g / kg, and continuously injecting for 21 days to complete molding. According to the invention, a method of increasing the use dosage of corticosterone and simultaneously adding estradiol (estrogen with the highest in-vivo content and strongest activity) is adopted, so that the modeling time can be shortened. The corticosterone and the estradiol are combined for tree shrew depression modeling, so that the modeling time can be effectively shortened, and the optimal modeling effect is achieved by adopting a special estradiol dosage.

Description

Technical Field

[0001] The invention belongs to the technical field of disease animal model construction, and in particular relates to a method for constructing a tree shrew depression model. Background Art

[0002] Depression is a mental illness characterized by low mood. Clinical manifestations include low mood, lack of energy, sleep disorders, slow thinking, reduced speech and movement, loss of interest, and suicidal tendencies in severe cases. Depression is a multifactorial disease with overlapping causal pathways. Its pathogenesis is still unclear and may involve many factors such as inflammatory immune system, monoamine neurotransmitters, neurogenesis, neuroplasticity, hippocampal ferroptosis, environment and genetics. At present, scientists have developed corresponding animal models, which reproduce the typical symptoms of human depression and can help us better understand the pathogenesis of the disease and then implement depression-related research. Due to the inherent limitations of human research and the unclear pathogenesis of depression, there are currently no clear experimental indicators, so the use of animal models is still a necessary step in identifying the neural mechanisms of depression and discovering new drugs and treatments. At present, the commonly used depression modeling animals are mainly rodents (rats, mice), and the modeling methods mainly include stress modeling, drug modeling, surgical modeling and genetic modeling.

[0003] Stress modeling methods, such as the behavioral despair model, simulate the occurrence and development of the main symptoms of human depression, but due to the complexity of immobility factors, the results may be affected by factors such as exercise fatigue and state adaptation, thereby reducing the effectiveness of the model; commonly used drug modeling, such as the reserpine-induced model, has a short modeling time, simple operation, and less animal pain, but its animal mortality rate is high, and the model's pathological process is inconsistent with individual depression. It is often used to screen effective therapeutic ingredients in traditional Chinese medicine and evaluate drug efficacy; the surgery itself may cause additional stress to the animals, and the animal mortality rate is high, and higher experimental technical capabilities are required; transgenic animal model depression is a multi-gene disease, and genes interact with environmental factors. Therefore, the depression model of a single gene mutation cannot fully reveal the pathological mechanism of depression, and the model is expensive, so its clinical application is limited.

[0004] The establishment of an animal model of depression can help researchers explore the pathogenesis of depression. In addition, the research on antidepressant drugs also requires the extensive use of animal models. At the same time, many models use certain behavioral manifestations of animals to simulate specific symptoms of depression, which is also of great significance for the research of specific symptoms. Therefore, creating an ideal animal model is one of the technical difficulties in overcoming depression research. Summary of the invention

[0005] The purpose of the present invention is to provide a method for modeling a tree shrew depression model. Tree shrews are a small animal closely related to primates, have a high brain-to-body mass ratio, a relatively developed nervous system and a stress system similar to that of humans, and therefore have irreplaceable advantages over other non-primate animal models.

[0006] The object of the present invention is achieved by comprising the steps of: (1) Dissolve 20 mg of corticosterone in 10 μl of dimethyl sulfoxide (DMSO) and dilute to 1 ml with saline; dissolve 100 mg of estradiol in 1 ml of dimethyl sulfoxide (DMSO) to prepare a 100 mg / ml estradiol stock solution; (2) Every afternoon between 14:00 and 16:00, the tree shrews were subcutaneously injected with the corticosterone solution prepared in step (1) at a volume of 27 μg / kg. One hour after the injection of the corticosterone solution, the estradiol stock solution prepared in step (1) was diluted with normal saline and then subcutaneously injected into the tree shrews at a volume of 50 μg / kg to 75 μg / kg. The injections were continued for 21 days to complete the modeling.

[0007] The beneficial effects of the present invention are: The present invention adopts a method of increasing the dosage of corticosterone and simultaneously adding estradiol (a type of estrogen with the highest content and the strongest activity in the body) to shorten the modeling time. The combination of corticosterone and estradiol for tree shrew depression modeling can effectively shorten the modeling time, and a special dosage of estradiol is used to achieve the best modeling effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The results of the autonomic activity scores of the tree shrew depression model induced by corticosterone in the first group of experiments; Figure 2 The results of the first group of experiments on sugar preference in the tree shrew depression model induced by corticosterone; Figure 3 This is the blood glucose measurement result of the first group of experiments in the tree shrew depression model induced by corticosterone; Figure 4 The results of the first group of experiments on 5-HT in the tree shrew depression model induced by corticosterone; Figure 5 This is the experimental result of the first group of experiments on the NE model of tree shrew depression induced by corticosterone; Figure 6 The results of the first group of experiments on the DA model of tree shrew depression induced by corticosterone; Figure 7 The results of the second group of experiments on the autonomic activity scores of the tree shrew depression model induced by corticosterone; Figure 8 The results of the second group of experiments on sugar preference in the corticosterone-induced depression model of tree shrews Fig. 9 The results of blood glucose measurement in the corticosterone-induced depression model of tree shrews in the second group of experiments; Fig.10 The results of the 5-HT experiment in the second group of experiments on the tree shrew depression model induced by corticosterone; Fig.11 The results of the second group of experiments on the NE model of tree shrew depression induced by corticosterone; Fig.12 These are the results of the second group of experiments on the DA model of tree shrew depression induced by corticosterone. DETAILED DESCRIPTION

[0009] The technical solution of the present invention will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of the embodiments. All other embodiments belong to the protection scope of the present invention.

[0010] A method for establishing a tree shrew depression model comprises the following steps: (1) dissolving 20 mg of corticosterone in 10 μl of DMSO and diluting the volume to 1 ml with physiological saline; dissolving 100 mg of estradiol in 1 ml of DMSO to prepare a 100 mg / ml estradiol stock solution; (2) Every day between 14:00 and 16:00, the tree shrews were subcutaneously injected with the corticosterone solution prepared in step (1) at a dose of 27 μg / kg. One hour after the injection of the corticosterone solution, the tree shrews were subcutaneously injected with the estradiol stock solution prepared in step (1) at a dose of 50 μg / kg to 75 μg / kg. The injections were continued for 21 days to complete the modeling.

[0011] The tree shrew is a common male Burmese tree shrew.

[0012] The tree shrew has a body mass of 120-150 g.

[0013] The tree shrews are kept at room temperature of 24±2°C, relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

[0014] Experiment 1 1.1 Experimental animals Fifteen ordinary male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24±2)℃, relative humidity (50±5)%, and light / dark cycle of 12h / 12h.

[0015] 1.2 Main Reagents

[0016] 1.3 Main instruments

[0017] 1.4 Preparation of drugs and reagents Corticosterone: fresh corticosterone solution was prepared every day, that is, 20 mg of corticosterone was dissolved in 10 μl of DMSO and the volume was adjusted to 1 ml with normal saline.

[0018] 1.5 Experimental methods 1.5.1 Animal model preparation Experimental groups: ①, normal group, ②, solvent group, ③, corticosterone group Group ③ was given subcutaneous injection of corticosterone (27 mg / kg) to tree shrews between 14:00-16:00 every afternoon for 21 consecutive days; Group ① was given an equal amount of normal saline at the same time, and Group ② was given an equal amount of normal saline containing 1% DMSO at the same time.

[0019] 1.5.2 Scoring of autonomous activities The autonomous activity of tree shrews was analyzed for 5 min before modeling (0d), 7d, 14d, 21d, 28d, and 35d. The autonomous activity score included movement (horizontal displacement) and jumping (vertical displacement), and the sum of the two times represented the relative activity of tree shrews in each group.

[0020] 1.5.3 Sugar water preference experiment Two days before the model, each tree shrew was given two bottles of 1% sucrose water at the same time. After 24 hours, one of the bottles was replaced with pure water for 12 hours. During this period, the position of the water bottle was changed every 2 hours. After the adaptation period, the tree shrews were deprived of food and water for 12 hours. Each tree shrew was given a bottle of 1% sucrose water and pure water, and the volume of water in the horizontal was recorded in advance. After 6 hours (9:00-15:00), the volume of the remaining sugar water and pure water was measured and recorded with a measuring cylinder. The percentage of sugar water preference was calculated according to the formula: Sugar water preference percentage = 1% sucrose drinking amount / (1% sucrose drinking amount + pure water drinking amount) × 100%.

[0021] The above experimental method can be used to obtain the sugar water preference percentage of tree shrews on day 0. The same method is used to conduct sugar water preference experiments on tree shrews on days 7, 14, 21, 28, and 35.

[0022] 1.5.4 Blood glucose measurement Blood samples were collected from tree shrews for blood glucose measurement before modeling (0d), 7d, 14d, 21d, 28d, and 35d.

[0023] 1.5.5 Elisa detection of 5-hydroxytryptamine (ST / 5-HT), norepinephrine (NA / NE), and dopamine (DA) Before modeling (0d), 7d, 14d, 21d, 28d, and 35d, blood was collected from tree shrews, and serum was separated after centrifugation. Elisa experiments were performed to detect the contents of 5-hydroxytryptamine (ST / 5-HT), norepinephrine (NA / NE), and dopamine (DA).

[0024] 1.6 Experimental Results 1.6.1 Scoring of autonomous activities The tree shrews in the normal group and the solvent group were very active on the 0th, 7th, 14th, 21st, 28th and 35th days, constantly turning upside down or moving horizontally in the cage; the tree shrews in the corticosterone group were basically the same as the normal group on the 0th, 7th, 14th and 21st days, and were very active. On the 28th and 35th days, their activity decreased, the number of jumps decreased, and the total score was lower than that of the normal group, especially on the 35th day, when their activity was the lowest.

[0025]

[0026] 1.6.2 Sugar water preference experiment The tree shrews in the normal group and the solvent group showed a significantly higher preference for sucrose water than for ordinary distilled water on days 0, 7, 14, 21, 28, and 35. Compared with the normal group, the corticosterone group showed little difference on days 0, 7, 14, and 21, but showed a lower preference for sucrose water on days 28 and 35, and it was more obvious on day 35.

[0027]

[0028] 1.6.3 Blood glucose measurement The blood glucose levels of tree shrews in the normal group and the solvent group were within the normal range on days 0, 7, 14, 21, 28, and 35. Compared with the normal group, the blood glucose levels of the corticosterone group showed an increasing trend on days 0, 7, 14, 21, 28, and 35, and exceeded the normal range on day 28.

[0029]

[0030] 1.6.4 5-hydroxytryptamine (5-HT) content Compared with the normal group, the 5-HT content in the solvent group was not much different, and the 5-HT content in the corticosterone group was basically the same on the 0th, 7th, 14th, and 21st days, but gradually decreased on the 28th and 35th days, and was more obvious on the 35th day.

[0031]

[0032] 1.6.5 Norepinephrine (NA / NE) content Compared with the normal group, the NE content in the solvent group was not much different, and the NE content in the corticosterone group was basically the same on days 0, 7, 14, and 21, but gradually decreased on days 28 and 35, and was more obvious on day 35.

[0033]

[0034] 1.6.6 Dopamine (DA) content Compared with the normal group, the DA content in the solvent group was not much different, and the DA content in the corticosterone group was basically the same on days 0, 7, 14, and 21, but gradually decreased on days 28 and 35, and was more obvious on day 35.

[0035]

[0036] Experiment 2 2.1 Experimental animals Thirty ordinary male Burmese tree shrews weighing 120-150 g were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK (Yunnan) K2023-0003] and kept in the ordinary animal room of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003] in stainless steel cages at room temperature (24±2)℃, relative humidity (50±5)%, and light / dark cycle of 12h / 12h.

[0037] 2.2 Main reagents

[0038] 2.3 Main instruments

[0039] 2.4 Preparation of drugs and reagents Corticosterone: Fresh corticosterone solution was prepared daily, i.e. 20 mg of corticosterone was dissolved in 10 μl of DMSO and the volume was adjusted to 1 ml with normal saline.

[0040] Estradiol: Dissolve 100 mg of estradiol in 1 ml of DMSO to prepare a 100 mg / ml estradiol stock solution. Dilute with saline before use and prepare it before use.

[0041] 2.5 Experimental methods 2.5.1 Animal model preparation Experimental groups: ①, control group, ②, corticosterone (27 mg / kg) group, ③, corticosterone (40 mg / kg) group, ④, corticosterone (27 mg / kg) + estradiol (25 μg / kg), ⑤, corticosterone (27 mg / kg) + estradiol (50 μg / kg), ⑥, corticosterone (27 mg / kg) + estradiol (75 μg / kg) Groups ②-⑥ were given subcutaneous injections of corticosterone according to the dosage between 14:00 and 16:00 every afternoon for 21 consecutive days. Groups ④-⑥ were given subcutaneous injections of estradiol according to the dosage 1 hour after the corticosterone injection for 21 consecutive days. Group ① was given an equal amount of saline containing 1% DMSO at the same time.

[0042] 2.5.2 Scoring of autonomous activities The autonomous activity of tree shrews was analyzed for 5 min before modeling (0d), 7d, 14d, and 21d. The autonomous activity score included movement (horizontal displacement) and jumping (vertical displacement), and the sum of the two times represented the relative activity of tree shrews in each group.

[0043] 2.5.3 Sugar water preference experiment Two days before the model, each tree shrew was given two bottles of 1% sucrose water at the same time. After 24 hours, one of the bottles was replaced with pure water for 12 hours. During this period, the position of the water bottle was changed every 2 hours. After the adaptation period, the tree shrews were deprived of food and water for 12 hours. Each tree shrew was given a bottle of 1% sucrose water and pure water, and the volume of water in the horizontal was recorded in advance. After 6 hours (9:00-15:00), the volume of the remaining sugar water and pure water was measured and recorded with a measuring cylinder. The percentage of sugar water preference was calculated according to the formula: Sugar water preference percentage = 1% sucrose drinking amount / (1% sucrose drinking amount + pure water drinking amount) × 100%.

[0044] The above experimental method can be used to obtain the sugar water preference percentage of tree shrews on day 0, and the same method is used to conduct sugar water preference experiments on days 7, 14, and 21 of tree shrews.

[0045] 2.5.4 Blood glucose measurement Blood samples were collected from tree shrews for blood glucose measurement before modeling (0d), 7d, 14d, and 21d.

[0046] 2.5.5 Elisa detection of 5-hydroxytryptamine (ST / 5-HT), norepinephrine (NA / NE), and dopamine (DA) content Before modeling (0d), 7d, 14d, and 21d, blood was collected from tree shrews, and serum was separated after centrifugation. Elisa experiments were performed to detect the content of 5-hydroxytryptamine (ST / 5-HT), norepinephrine (NA / NE), and dopamine (DA).

[0047] 2.6 Experimental Results 2.6.1 Scoring of autonomous activities All groups of tree shrews were very active before the experiment (day 0), constantly flipping up and down or moving horizontally in the cage. The tree shrews in the control group were very active throughout the experiment. Compared with the control group, the tree shrews in the corticosterone (27 mg / kg) and corticosterone (40 mg / kg) groups showed a slight decrease in the scores of spontaneous activity on the 21st day, which was consistent with the previous results; the tree shrews in the corticosterone + estradiol group began to show a decrease in spontaneous activity scores on the 7th day, and the degree of decrease in spontaneous activity scores of 50 μg / kg and 75 μg / kg estradiol was more obvious, but the difference between the two was not large.

[0048]

[0049] 2.6.2 Sugar water preference experiment Before the experiment (day 0), tree shrews in all groups preferred sucrose water more than ordinary distilled water. Tree shrews in the control group maintained their preference for sucrose water throughout the experiment. Compared with the control group, the percentage of tree shrews' preference for sucrose water in the corticosterone (27 mg / kg) and corticosterone (40 mg / kg) groups decreased slightly on day 21, which was consistent with the previous results. The percentage of tree shrews' preference for sucrose water in the corticosterone + estradiol group decreased from day 7, and the percentage of preference for 50 μg / kg and 75 μg / kg estradiol sucrose water decreased more significantly, but the difference between the two was not large.

[0050]

[0051] 2.6.3 Blood glucose measurement The blood glucose of tree shrews in all groups was within the normal range before the experiment (day 0), and the blood glucose of tree shrews in the control group changed little during the whole experiment. Compared with the control group, the blood glucose of tree shrews in the corticosterone (27 mg / kg) group was within the normal range throughout the experiment, but showed an upward trend, which was consistent with the previous results; the blood glucose of tree shrews in the corticosterone (40 mg / kg) group increased more significantly than that in the corticosterone (27 mg / kg) group, and exceeded the normal range at day 21; the blood glucose of tree shrews in the corticosterone + estradiol group remained within the normal range throughout the experiment.

[0052]

[0053] 2.6.4 5-hydroxytryptamine (5-HT) content Compared with the control group, the 5-HT content of tree shrews in the corticosterone (27 mg / kg) group and the corticosterone (40 mg / kg) group did not change much, except for a slight downward trend on the 21st day; the 5-HT content of tree shrews in the corticosterone + estradiol group decreased on the 7th day, and the decrease in 50μg / kg and 75μg / kg estradiol 5-HT content was more significant, and reached the minimum on the 21st day, but the difference between the two was not large.

[0054]

[0055] 2.6.5 Norepinephrine (NA / NE) content Compared with the control group, the NE content of tree shrews in the corticosterone (27 mg / kg) group and the corticosterone (40 mg / kg) group did not change much, except for a slight downward trend on the 21st day; the NE content of tree shrews in the corticosterone + estradiol group decreased on the 7th day, and the decrease in NE content of 50 μg / kg and 75 μg / kg estradiol was more significant, and reached the minimum on the 21st day, but the difference between the two was not large.

[0056]

[0057] 2.6.6 Dopamine (DA) content Compared with the control group, the DA content of tree shrews in the corticosterone (27 mg / kg) group and the corticosterone (40 mg / kg) group did not change much, except for a slight downward trend on the 21st day; the DA content of tree shrews in the corticosterone + estradiol group decreased on the 7th day, and the decrease in 50 μg / kg and 75 μg / kg estradiol DA content was more significant, and reached the minimum on the 21st day, but the difference between the two was not large.

[0058]

Claims

1. A method for establishing a tree shrew depression model, characterized in that: The steps include: (1) Dissolve 20 mg of corticosterone in 10 μl of DMSO and dilute to 1 ml with saline. Dissolve 100 mg of estradiol in 1 ml of DMSO to prepare a 100 mg / ml estradiol stock solution. (2) Every day between 14:00 and 16:00, the tree shrews were subcutaneously injected with the corticosterone solution prepared in step (1) at a dose of 27 μg / kg. One hour after the injection of the corticosterone solution, the tree shrews were subcutaneously injected with the estradiol stock solution prepared in step (1) at a dose of 50 μg / kg to 75 μg / kg. The injections were continued for 21 days to complete the modeling.

2. The method for establishing a tree shrew depression model according to claim 1, characterized in that: The tree shrew is a common male Burmese tree shrew.

3. The method for establishing a tree shrew depression model according to claim 1, characterized in that: The tree shrew has a body mass of 120-150 g.

4. The method for establishing a tree shrew depression model according to claim 1, characterized in that: The tree shrews are kept at room temperature of 24±2°C, relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

Images