CN112106729A - Modeling method for mice with depression, liver depression and spleen deficiency syndrome - Google Patents
Modeling method for mice with depression, liver depression and spleen deficiency syndrome Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
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- A—HUMAN NECESSITIES
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- A01K2207/00—Modified animals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
A modeling method of a mouse with depression, liver depression and spleen deficiency diseases relates to the technical field of depression research, and 28-day continuous stimulation is performed on healthy mice, each stimulation is used for 4 times on average, and the stimulation types are as follows: any one of 24-hour stimulation of water and food deprivation, 1-minute stimulation of tail clamping, 5-minute stimulation of ice water swimming at 10 ℃, 5-minute stimulation of horizontal shaking with frequency of 1 time/s, 24-hour stimulation of light irradiation, 24-hour stimulation of saturated water-wet padding or 24-hour stimulation of inclined squirrel cage with 45 degrees; from day 15 to day 28 of the above continuous stimulation, mice were also gavaged with 0.5mL of an aqueous suspension of rhubarb powder at a concentration of 15% (wt) per day. The obtained model mouse has the symptomatic expression of depression liver-stagnation and spleen-deficiency syndrome, and can be used for further research on pathogenesis and treatment of depression liver-stagnation and spleen-deficiency syndrome.
Description
Technical Field
The invention relates to the technical field of depression research, in particular to a molding technology of animals with diseases.
Background
The depression is a serious affective disorder, and researches find that the prevalence rate of the depression is as high as about 10 percent and the lifetime prevalence rate of the depression is as high as 20 percent all over the world; the suicide rate of the depression is high, more than half of depression patients have suicide thoughts, and the number of deaths caused by the suicide of the depression per year exceeds 100 ten thousand. High morbidity, disability rate and suicide rate bring serious influence to families and society. Moreover, the incidence of depression tends to increase year by year.
The curative effect of the treatment of the depression is poor. Only less than 50% of patients benefit from standard antidepressant therapy, and around 70% remain with some symptoms. This may be caused by the fact that depression is a highly heterogeneous disease, the diagnosis and treatment process ignores the difference of symptoms, the clinical symptoms of depression are greatly different from one patient to another, and the difference of symptoms affects the treatment outcome. In the study of 853 symptoms of depression patients, different combinations of 119 symptoms are found, which indicates that the depression is not a single disease process but a systemic disease involving multiple systems and multiple organs, the pathological process of the depression is very complex, and the depression relates to inflammatory reaction, endocrine metabolism disorder, oxidative stress, neurotransmitter abnormality and the like, however, the treatment effect of the depression is not ideal, and the diagnosis and treatment individuation and the identification of unique symptoms are related to prognosis. The differentiation of syndromes according to traditional Chinese medicine is the process of differentiating diagnosis and treatment by combining various information such as physical signs, symptoms, pulse conditions, tongue conditions and the like, and researches show that diseases with different syndrome types need to be treated in a targeted manner according to individual differences.
The principle of traditional medicine "treatment based on syndrome differentiation" emphasizes the individual differences of diseases. Liver depression and spleen deficiency, deficiency of both heart and spleen, qi stagnation and blood stasis, liver depression and qi stagnation, disharmony between heart and kidney, and liver depression and phlegm stagnation are six main traditional Chinese medicine symptoms of depression, wherein the occurrence frequency of the liver depression and spleen deficiency syndrome is the highest. Epidemiological investigation carried out by nobility and the like shows that the diagnosis standard of the depression liver depression spleen deficiency syndrome is as follows: depressed mood, pessimistic and boredom, good sigh, listlessness, debilitation, anorexia, emaciation, and thready pulse, such as four of the seven items, namely liver depression two items (essential for depressed mood) and spleen deficiency two items. Therefore, the development trend of precise medical treatment is to provide individual treatment according with the characteristics of the disease and syndrome for depression patients by combining different syndrome types of traditional Chinese medicine.
The animal model is an important means for researching the pathogenesis of depression, and the research on a disease model combining depression and disease symptoms is rarely carried out at present. At present, the model building method of depression is mostly adopted by animal models related to different traditional Chinese medicine syndrome types of depression, namely the model building mode of western medicine 'disease', the traditional Chinese medicine syndrome types are summarized by observation or possible syndrome types are deduced reversely according to the treatment effect of traditional Chinese medicines, hysteresis exists and specificity is lacked, so that the inconsistency and uncertainty of research results are caused, and the clinical transformation of basic research is influenced.
The disease animal model with good credibility and validity is an important guarantee that research results can be smoothly transformed from a laboratory to clinic. The animal model of depression is constructed according to etiology and pathology in western medicine research, the credibility and the validity are high, but the individual difference of animals is ignored, the construction of the disease model is emphasized in the modeling process, the construction of the syndrome model is weakened, and the disease and the animal model are relatively ideal animal models at present.
Disclosure of Invention
Based on the theoretical principle of 'disease and syndrome combination', the invention aims to overcome the defects of the traditional animal model of depression liver depression and spleen deficiency, and the modeling method of the depression liver depression and spleen deficiency disease mouse is stable, high in confidence and efficacy and used for basic research.
The technical scheme of the invention is as follows: healthy mice were given 28 days of continuous stimulation, with an average of 4 per stimulation, of the type: any one of 24-hour stimulation of water and food deprivation, 1-minute stimulation of tail clamping, 5-minute stimulation of ice water swimming at 10 ℃, 5-minute stimulation of horizontal shaking with frequency of 1 time/s, 24-hour stimulation of light irradiation, 24-hour stimulation of saturated water-wet padding or 24-hour stimulation of inclined squirrel cage with 45 degrees; from day 15 to day 28 of the above continuous stimulation, mice were also gavaged with 0.5mL of an aqueous suspension of rhubarb powder at a concentration of 15% (wt) per day.
Based on the theory of 'combination of diseases and symptoms', the invention establishes a depression model by using CUMS according to the chronic unpredictable mild stress and the rheum officinale gavage method, and simultaneously establishes a liver-stagnation and spleen-deficiency syndrome model by using the rheum officinale gavage method, and the newly established model needs to have the expression of depression and the liver-stagnation and spleen-deficiency syndrome at the same time.
At present, the mechanism of CUMS which can cause the mouse depression-like behavior is a 'predicament' in a simulated real life, and the mouse is in chronic stress for a long time without the principle of applying the mouse, namely, the mouse is subjected to aversive stimulation which cannot be predicted and cannot escape for a long time, and becomes desperate gradually, and the behavior is reduced, dullness, excitability and cognitive function are reduced. The molding effect can be evaluated by measuring the behavior thereof.
The above chronic unpredictable mild stress model is one of the classic animal models of depression, which is the exposure of an animal to sustained mild unpredictable stimuli during the molding process, inducing the animal to produce behavior similar to that of a depressed patient. The rhubarb stomach-filling method is a classic method for constructing the syndrome of liver depression and spleen deficiency by performing stomach filling on experimental animals by using a rhubarb water decoction or a rhubarb powder suspension to simulate the pathological evolution process of bitter cold, stomach failure, long-term liver depression, wood depression with soil and liver depression and spleen deficiency.
The rhubarb powder is used for intragastric administration from day 15, aims to superpose spleen deficiency symptoms on the basis of depression, so that a complete depression liver depression spleen deficiency model is established, and the model building effect is also evaluated through the behavior of model animals.
Through the technical method, the obtained model mouse shows typical depression symptoms of slow activity speed, reduced activity, reduced sucrose preference index, prolonged tail suspension immobility time and prolonged forced swimming immobility time; meanwhile, the model mouse has the symptoms of poor appetite, slow weight increase and loose stool due to liver depression and spleen deficiency, which indicates that the model mouse has the symptomatic expression of depression and the syndrome of liver depression and spleen deficiency. The disease model mouse has the clinical phenotype of successfully simulated depression diseases and liver depression and spleen deficiency, and accords with the symptoms of mental stress, depression and anger, liver qi depression, liver loss and catharsis and transverse adverse attack on the spleen; or the pathogenesis of the spleen impairment due to thinking, the deficiency of earth and the wood, the dysfunction of the spleen in transport and the dysfunction of qi movement in ascending and descending can be used for further research on the pathogenesis and treatment of the depression with liver depression and spleen deficiency.
In order to ensure that the total weight of each gavage of a model mouse (about 20 g) is 75mg (comparing 50mg, selecting after 75mg and 100mg effects), the concentration of the aqueous suspension of the rhubarb powder adopted by the invention is 15% (wt), the total intake is 0.5ml (the maximum volume of the gavage of the mouse is 0.8 ml), the stomach gavage amount of the mouse can influence the comfort level and the activity of the mouse, in order to avoid the influence of the stomach gavage amount on the model mouse, the invention separately gavage the stomach in the morning, evening and three times every day when the aqueous suspension of the rhubarb powder is gavage, and the stomach gavage amount of each time is respectively: 0.2mL, 0.15mL and 0.15mL (the sequence can be random), which not only ensures the amount of rhubarb powder needed for the syndrome of liver depression and spleen deficiency, but also avoids the influence of irrelevant stimulation on a disease model.
With 15 watt LED cold light, illumination intensity is 15 ~ 20lx, and the irradiation distance is 50cm, simulation daytime light, and the model mouse that shines lasts 24 hours is amazing as light irradiation, disturbs the mouse sleep rhythm, makes its irritability, easily irritates, appears the overfatigue, the irregular phenomenon of sleep, the influence of sleep to the mood in the simulation reality.
The padding (such as wood shavings) is preferably soaked completely in room-temperature water for 24h stimulation, and the padding can not be soaked in too little water, so that the 'escape' behavior of a model mouse in the molding process can be caused, and the molding effect can not be achieved; too much water will cause the mice to be fully soaked in water for 24h, and the cold and water soaking cause additional stimulation to the model and interfere with the modeling effect.
In order to avoid the influence of independent variables such as age, weight, sex and environment on animals, random variables are selected for homogenization, and a method for controlling the independent variables ensures the influence of other factors on animal models with diseases. The healthy mouse is obtained after an open field experiment, a cane sugar water preference experiment, a tail suspension experiment and a forced swimming experiment are completed after an ICR adult male mouse which is born for 6-8 weeks and has the weight of 25-29g is bred for one week in an animal room with the environmental temperature of 20-26 ℃ and the humidity of 40-60%.
Detailed Description
Firstly, a molding process:
1. preparation of experimental animals:
30 adult ICR male mice (animal production license: SCXK (su) 2017-. The animal house is bred adaptively for one week in a Chinese and western medicine combined system experiment of Yangzhou university, the environmental temperature of the animal house is 20-26 ℃, and the humidity is 40-60%.
After the adaptive feeding is finished, the measurement of the baseline body weight, the open field experiment, the cane sugar water preference experiment, the tail suspension experiment, the forced swimming experiment and the excrement character are completed.
The mice were then divided into 15 control groups and 15 experimental groups. In order to avoid mutual biting among the mice in the molding process, only 1 mouse is raised in each cage.
The experimental group was modeled by a combination of disease and syndrome, and the control group was not treated.
The raising environment and mode of the experimental group and the control group are completely consistent except the stimulation of the molding behavior.
2. Modeling of depression:
the experimental groups were subjected to 28 days of continuous stimulation, with an average of 4 times per stimulation, and 1 randomly selected from the following 7 types per day, with reference to a chronic unpredictable mild stress model.
The water and food are forbidden for 24 hours.
② clamping tail for 1 min.
③ swimming for 5min with 10 ℃ ice water.
Mixing normal temperature water and ice at volume ratio of 2: 1, standing at room temperature, and swimming for 5min when temperature is reduced to 10 deg.C.
Fourthly, horizontally shaking for 1 time/s for 5 min.
Light stimulation for 24 h.
Light source requirements: the 15W LED has the cold light, the illumination intensity is 15-20 lx, and the irradiation distance is 50 cm.
Sixthly, wetting the padding for 24 hours.
Saturated water-wet padding (e.g., wood shavings) fully saturated with room temperature water was placed in the mouse cage, and the test rats were placed in the mouse cage for 24 hours.
And the mouse cage is inclined by 45 degrees for 24 hours.
Note that the above water and food deprivation may not be used continuously on the same mouse, and specifically the following schedule may be adopted:
schedule schedule table
| Time of day | Experimental group | Control group | Time of day | Experimental group | Control group |
| Day 1 | Clip tail | / | Day 15 | Gavage with inclined mouse cage and rhubarb | / |
| Day 2 | Water and food deprivation | / | Day 16 | No water and no food, and radix Et rhizoma Rhei for intragastric administration | / |
| Day 3 | Horizontal shaking | / | Day 17 | Wet padding and rhubarb for intragastric administration | / |
| Day 4 | Swimming with ice water | / | Day 18 | Gavage with clip tail and rhubarb | / |
| Day 5 | Light stimulation | / | Day 19 | Horizontal shaking and rheum officinale intragastric administration | / |
| Day 6 | Wet padding | / | Day 20 | Ice water swimming and rhubarb drenching | / |
| Day 7 | Inclined mouse cage | / | Day 21 | Light stimulation and rheum officinale lavage | / |
| Day 8 | Clip tail | / | Day 22 | Gavage with inclined mouse cage and rhubarb | / |
| Day 9 | Horizontal shaking | / | Day 23 | No water and no food, and radix Et rhizoma Rhei for intragastric administration | / |
| Day 10 | Swimming with ice water | / | Day 24 | Gavage with inclined mouse cage and rhubarb | / |
| Day 11 | Light stimulation | / | Day 25 | Wet padding and rhubarb for intragastric administration | / |
| Day 12 | Water and food deprivation | / | Day 26 | Light stimulation and rheum officinale lavage | / |
| Day 13 | Wet padding | / | Day 27 | Gavage with clip tail and rhubarb | / |
| Day 14 | Horizontal shaking | / | Day 28 | Ice water swimming and rhubarb drenching | / |
3. Modeling of liver depression and spleen deficiency syndrome:
according to the model making mode of liver depression and spleen deficiency, slightly adjusting, beginning on the 15 th day of chronic unpredictable stress model making in an experimental group for 14 consecutive days, and simultaneously irrigating 0.5mL of rhubarb powder aqueous suspension with the stomach concentration of 15% (wt) to an experimental mouse every day, wherein the injection is divided into three times in the morning, at the noon, at the evening, 0.2mL in the morning, 0.15mL in the middle and at the evening and 0.15mL in the evening (also: 0.15mL in the morning, 0.2mL in the middle and at the evening, 0.15mL in the morning, 0.15mL in the middle and at the evening and 0.2mL in the evening).
4. Observation indexes and methods:
and 2d before molding and 2d after molding are carried out to complete the weight measurement, behavior experiment and feces collection of two groups of mice, and in order to avoid the influence of the sequence on the observation indexes, the experimental group and the control group are carried out in a crossed manner.
5. And (3) verifying the credibility of the model:
the difference between the model mouse and the control group reflects the effectiveness of the model, and the phenotypic index of the model mouse reflects the reliability of the model.
II, evaluation basis and index:
1. indices of depression:
open field experiments, sucrose preference experiments, tail suspension experiments and forced swimming experiments are commonly used indexes for evaluating depression models of experimental animals.
The test evaluates the degree of depression of model mice in terms of liveness and average speed in open field experiments, sucrose preference index, tail suspension and forced swimming immobility time.
2. Indices of liver depression and spleen deficiency syndrome:
bristol Stool Form Scale (BSFS) is often used to assess the corresponding Stool trait and thus the severity of the pattern of liver depression and spleen deficiency.
The study used mouse body weight and fecal characteristics as indicators for assessing liver depression and spleen deficiency.
The mice were placed in an observation box at noon one day before and one day after the end of the experiment, the first feces were collected, and classified and evaluated according to the classification standard of BSFS and 1-7 scores of types 1-7 of feces classification, as shown in the following table.
| Typing | Traits | Score value |
| Class 1 | Hard and dry stool and granular | 1 minute (1) |
| Class 2 | Hard and dry stool and strip | 2 is divided into |
| Class 3 | Uneven and slightly dry surface | 3 points of |
| Class 4 | Strip-shaped soft stool with smooth surface | 4 is divided into |
| Class 5 | Soft stool and urine | 5 points of |
| Class 6 | Thin and rotten stool and unformed stool | 6 minutes |
| Class 7 | Water sample toilet | 7 points of |
Thirdly, analyzing results:
1. the modeling reduces the average speed and the activeness of the mouse, the activity of the mouse is limited, the preference index of cane sugar is reduced, the quick feeling is lost, the immobility time in a tail suspension experiment is prolonged, the struggling time is prolonged during forced swimming, and the hopeless depressive state is presented.
Compared with the prior art of the mold of the group,▲ P＜0.05，▲▲ Pless than 0.01; compared with the control group after the model is made,## P＜0.01。
note: in the above table▲And▲▲the model mouse shows obvious depression performance after model making compared with that before model making, the activity speed and the activity degree are both reduced, the intake of sugar water is reduced, the immobility time is prolonged during tail suspension and forced swimming, and the control group mouse has no change, and transverse comparison shows the model making effect.
In the above table##Shows that the model mouse has obvious depression performance after model making compared with a control group mouse (without various stimulations), the activity speed and the activity degree are both reduced, the intake of sugar water is reduced, the immobility time is prolonged during tail suspension and forced swimming, and the longitudinal comparison shows the model making effect.
2. The modeling reduces the amplitude of the weight gain of the mice; the water content of the excrement of the mice is increased, and loose stools appear, which is similar to the appearance of liver depression and spleen deficiency.
Compared with the prior art of the mold of the group,▲ P＜0.05，▲▲ Pless than 0.01; compared with the control group after the model is made,# P＜0.05，## P＜0.01。
note: weight column in the upper table▲▲Shows that the weight average of the body of the model mouse and the control group mouse after molding is increased compared with that before molding,##the weight of the model mouse after molding is lower than that of the control group mouse, and the longitudinal comparison shows the molding effect.
BSFS column in the above table▲▲The water content of the excrement of the model mouse after the model is made is obviously higher than that of the excrement before the model is made, and the longitudinal comparison shows the model making effect;##the water content of the feces after model mouse molding is obviously increased compared with the feces of the control group mouse (without various stimulations), and the longitudinal comparison shows the molding effect.
By combining the characteristics of the diseases and the symptoms, the invention explores a modeling method of the depression liver-depression spleen-deficiency syndrome which accords with the clinical syndrome type. According to the modern discipline theory and the scientific knowledge of relevant experimental animals, the disease is copied to the animal body by using the modern medicine, and the 'syndrome' of the traditional Chinese medicine is copied to the animal model by using the factors such as the etiology and the like. The purpose of doing so is to make the "disease and syndrome combined" animal model not only have the characteristics of western medicine diseases, but also have the characteristics of traditional Chinese medicine syndromes, provide a research basis for individualized and accurate medical treatment, and be favorable for more comprehensively and intuitively referring to the traditional Chinese medicine clinical to carry out scientific research and the experimental results can be applied clinically in time.
Claims (5)
1. The molding method of the mouse with depression, liver depression and spleen deficiency diseases is characterized in that: healthy mice were given 28 days of continuous stimulation, with an average of 4 per stimulation, of the type: any one of 24-hour stimulation of water and food deprivation, 1-minute stimulation of tail clamping, 5-minute stimulation of ice water swimming at 10 ℃, 5-minute stimulation of horizontal shaking with frequency of 1 time/s, 24-hour stimulation of light irradiation, 24-hour stimulation of saturated water-wet padding or 24-hour stimulation of inclined squirrel cage with 45 degrees; from day 15 to day 28 of the above continuous stimulation, mice were also gavaged with 0.5mL of an aqueous suspension of rhubarb powder at a concentration of 15% (wt) per day.
2. The molding method according to claim 1, wherein: when the rhubarb powder aqueous suspension is perfused, the stomach is separately perfused three times in the morning, at noon and at night every day, and the stomach perfusion amount is respectively: 0.2mL, 0.15 mL.
3. The molding method according to claim 1 or 2, wherein: the light source of the light stimulation is 15 watt LED cold light, and the illumination intensity is 15-20 lx.
4. The molding method according to claim 1 or 2, wherein: the saturated water-wet padding is a squirrel cage padding which is completely soaked by room-temperature water.
5. The molding method according to claim 1, wherein: the healthy mice are obtained after an open field experiment, a cane sugar water preference experiment, a tail suspension experiment and a forced swimming experiment are completed after adult male ICR mice which are born for 6-8 weeks and have the weight of 25-29g are bred for one week in an animal room with the ambient temperature of 20-26 ℃ and the humidity of 40-60%.
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