CN111436396A - Method for establishing rat fear stress and gaseous pollutant composite exposure model - Google Patents
Method for establishing rat fear stress and gaseous pollutant composite exposure model Download PDFInfo
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- CN111436396A CN111436396A CN202010334604.5A CN202010334604A CN111436396A CN 111436396 A CN111436396 A CN 111436396A CN 202010334604 A CN202010334604 A CN 202010334604A CN 111436396 A CN111436396 A CN 111436396A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Abstract
The invention belongs to the technical field of animal model establishment, and discloses a method for establishing a rat fear stress and gaseous pollutant composite exposure model. The method comprises the following steps: (1) establishing a fear model: selecting 180-220g healthy male wistar rats, placing the rats in a closed environment, giving a 75dB 1000Hz sound signal, giving 1mA plantar current stimulation at the same time for 3s at the tail of the sound signal, repeating the cycle for 20 times every day, and normally feeding free edible water in the rest time; lasting for six weeks; (2) establishing a fear stress and gaseous pollutant composite exposure model: sucking gaseous pollutants into the rat which is built by the fear model, wherein the sucking time is 4 hours every day and lasts for 30 days; (3) and (5) sampling and detecting. The method of the invention combines two harmful factors and evaluates the toxicity hazard of human body.
Description
Technical Field
The invention relates to the technical field of animal model establishment, in particular to a method for establishing a rat fear stress and gaseous pollutant composite exposure model.
Background
The description of the background of the invention pertaining to the related art to which this invention pertains is given for the purpose of illustration and understanding only of the summary of the invention and is not to be construed as an admission that the applicant is explicitly or implicitly admitted to be prior art to the date of filing this application as first filed with this invention.
Various physical and chemical harmful factors in the closed space of the submarine cabin are various and exist at the same time. The influence of different degrees on all aspects can be caused to the sailor, the change of physiological indexes and the appearance of psychological problems can be caused, and various diseases can be caused.
Harmful gas in submarine operation environment is generated from nonmetal materials used by submarines in large quantity, cooking food, self metabolites of the submariners, machine operation and the like, such as carbon monoxide, dimethylbenzene, acrolein, hydrogen sulfide and the like.
The submarine crew is in danger of operation in a closed space in deep sea, various training and sudden conditions inside and outside the submarine when performing tasks, particularly long-time deep diving tasks, and psychological loads of fear stress states exist, and physical and psychological influences are generated by the coexistence of other harmful factors.
Disclosure of Invention
The invention aims to provide a method for establishing a rat fear stress and gaseous pollutant compound exposure model.
A method for establishing a rat fear stress and gaseous pollutant compound exposure model, which comprises the following steps:
(1) establishing a fear model: selecting 180-220g healthy male wistar rats, placing the rats in a closed environment, giving a 75dB 1000Hz sound signal, giving 1mA plantar current stimulation at the same time for 3s at the tail of the sound signal, repeating the cycle for 20 times every day, and normally feeding free edible water in the rest time; lasting for six weeks;
(2) establishing a fear stress and gaseous pollutant composite exposure model: sucking gaseous pollutants into the rat which is built by the fear model, wherein the sucking time is 4 hours every day and lasts for 30 days;
(3) and (5) sampling and detecting.
Further, the gaseous contaminant is xylene.
Further, the concentration of xylene inhaled by rats was 0.38 ppm.
Further, the concentration of xylene inhaled by rats was 3.8 ppm.
Further, the sampling test includes general behaviors, percent freezing time, central nervous system, immune system and endocrine system.
The invention has the following beneficial effects:
the method builds a stable and long-acting conditioned reaction fear model, realizes the compounding of two harmful factors by correlating the contamination and fear through specific conditioned signals, compounds the fear stress and the main gaseous pollutants in the submarine closed space working environment, researches the influence of harmful gas inhalation on the health conditions of animal physiological behaviors and the like under the fear stress state, and further discusses the evaluation of the toxicity hazard to human bodies.
Drawings
FIG. 1 is a graph of percent freezing time (FT%) for control group C versus model group F in accordance with the present invention;
FIG. 2 is a graph showing the weight gain of the control group C and the model group F according to the present invention;
FIG. 3 is a graph showing a comparison between the trajectory display and exploration behaviors of a control group C and a model group F according to the present invention;
FIG. 4 is a graph showing the results of the test of the effect of fear stress on the body according to the present invention;
FIG. 5 is a graph showing the results of the test of the effect of fear stress on the body according to the present invention;
FIG. 6 is a graph showing the results of the test of the effect of fear stress on the body according to the present invention;
FIG. 7 is a graph showing the results of the test of the effect of fear stress on the body according to the present invention;
FIG. 8 is a graph showing the results of measurement of the influence of fear stress on arachidonic acid metabolism according to the present invention;
FIG. 9 is a graph showing the results of pathological examination of hippocampus in accordance with the present invention;
FIG. 10 is a diagram showing the results of pathological examination of cerebral cortex according to the present invention;
FIG. 11 is a diagram showing the results of the amygdala assay in the brain of the present invention;
FIG. 12 is a graph showing the results of pharmacological tests of the present invention;
FIG. 13 is a graph showing the results of pathological liver tests according to the present invention;
FIG. 14 is a graph showing the results of measurement of Corticotropin Releasing Hormone (CRH) in blood according to the present invention;
FIG. 15 is a graph showing the results of detection of Corticosterone (CORT) in blood according to the present invention;
FIG. 16 is a graph showing the results of measurement of adrenocorticotropic hormone (ACTH) in blood according to the present invention;
FIG. 17 is a graph showing the results of the Norepinephrine (NE) detection in blood according to the present invention;
FIG. 18 is a graph showing the result of detecting Acetylcholine (ACH) in brain tissue according to the present invention;
FIG. 19 is a graph showing the result of Dopamine (DA) detection in brain tissue according to the present invention;
FIG. 20 is a graph showing the results of 5-hydroxytryptamine (5-HT) assay in brain tissue according to the present invention.
Detailed Description
The present application is further described below with reference to examples.
In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art.
A method for establishing a rat fear stress and gaseous pollutant compound exposure model, which comprises the following steps:
(1) establishing a fear model: selecting 180-220g healthy male wistar rats, placing the rats in a closed environment, giving a 75dB 1000Hz sound signal, giving 1mA plantar current stimulation at the same time for 3s at the tail of the sound signal, repeating the cycle for 20 times every day, and normally feeding free edible water in the rest time; lasting for six weeks;
(2) establishing a fear stress and gaseous pollutant composite exposure model: sucking gaseous pollutants into the rat which is built by the fear model, wherein the sucking time is 4 hours every day and lasts for 30 days;
(3) and (5) sampling and detecting.
The first stage uses fear experimental box to make the mould, is in relative confined environment with the experimental animal, gives the sound signal of specific frequency, and the vola electro photoluminescence is given simultaneously to audio frequency end several seconds, makes the animal produce the fear state. The animal produces the fear of condition reaction in the molding process, and this kind of specific audio frequency is the condition that the induced animal appears the fear state, hears this audio frequency promptly and will produce the fear state. Through long-time modeling experiments, the conditioned reflex is reinforced, and relatively long-time memory is formed, so that the time of the next compound infection experiment is facilitated.
In some embodiments of the invention, the gaseous contaminant is xylene.
In some embodiments of the invention, the concentration of xylene inhaled by rats is 0.38 ppm.
In some embodiments of the invention, the concentration of xylene inhaled by rats is 3.8 ppm.
In some embodiments of the invention, the sampling test includes general behavior, percent freezing time, central nervous system, immune system, and endocrine system.
Example 1
A method for establishing a rat fear stress and gaseous pollutant compound exposure model, which comprises the following steps:
60 SPF grade 130-.
The process is as follows:
180-220g healthy male wistar rats are randomly divided into a fear model group (F) and a control group (C), the tail parts are marked with numbers, and the weight is recorded. Planning a test flow according to the test conditions, and setting an instrument program template. The group F was given a 75dB 1000Hz sound signal, 3s at the end of the sound signal and 1mA plantar current stimulation, and the group C was given no electrical stimulation only with the sound signal, and the cycle was repeated 20 times daily for each group. And (5) normally feeding free water in the rest time. The test continued until fear status stabilized in group F animals. The instrument records data such as motion tracks, freezing time and the like. After the expected fear state is reached, the fear memory fading experiment is continued according to the duration of 4h of the compound gaseous pollutant required by the study (the period is a program of 20min, the cycle is 12 times, each time interval is 4h/d, and only the sound signal has no electrical stimulation). Treating animals and sampling.
The specific process is as follows:
the animals were randomly grouped, wherein 56 experimental animals were divided into 8 fear complex high concentration toxicant exposure groups (FH), 8 high concentration toxicant exposure groups (CH), 8 fear complex low concentration toxicant exposure groups (F L), 8 low concentration toxicant exposure groups (C L), 8 fear non-toxicant exposure groups (FC), 8 blank control groups (C), 8 fear complex low concentration toxicant exposure groups (F) and the remaining 4 prepared blank controls, and were raised in cages, 4 animals were housed in each cage, the cage numbers were FH1/FH2/CH1/CH2/F L1/F L2/C L1/C L2 FC 2/FC 1/2/C1/C2/F1/F2/C, and the animal tail markers were grouped and numbered.
Numbering a modeling method, experimental equipment and experimental process of the conditional fear model with the F experimental animal: a75 dB 1000Hz sound signal was administered, 1mA plantar current stimulation was simultaneously administered 3s at the end of the sound signal, and group C was given no electrical stimulation but the sound signal, and the cycle was repeated 20 times daily for each group. And (4) normally feeding the animals with free water for the rest of time until the fear state of the animals in the group F is stable.
After the modeling was completed, the model animal experiment of conditioned fear state complex xylene inhalation was continued. The concentration of dimethylbenzene is set to be 3.8ppm and 0.38ppm, and the toxicant exposure with the same concentration is divided into a fear toxicant exposure group and a contrast toxicant exposure group.
The kit respectively corresponds to 8 fear complex high-concentration toxicant exposure groups (FH)8, 8 high-concentration toxicant exposure groups (CH)8, 8 fear complex low-concentration toxicant exposure groups (F L) 8 and 8 low-concentration toxicant exposure groups (C L). the corresponding cage number is FH1/FH2/CH1/CH2/F L1/F L2/C L1/C L2.
Conditional fear experiment system (Beijing Zhongguo Dichu science and technology Limited ZS-KJ, containing L abMaze animal behavior analysis software and video and computer), xylene gas (Wuhan Nuruide specialty gas Limited, 450ppm in 10L tank, 0.7MPa), animal gas toxicant exposure instrument (Beijing Huilong and science and technology Limited, HRH-CSED-K), pump-suction xylene detector (Zhengzhou Junda instruments Limited, JA908), mini-speaker (playing manufactured audio file: 19min40s at 20min per week, beginning to last 20s of 75dB:1000Hz sound signal until the end of the cycle, 12 cycles, total 4h), mini-fan (used for air flow in the toxicant exposure box to improve environment), charge pool (used for backup power supply of detector, mini-speaker, mini-fan).
The contamination process is as follows:
four cages per batch of 16 experimental animals were used in concentration and groups for ambient xylene inhalation using a small animal exposure cabinet.
The experimental time of each batch is 4h, and the high-low concentration groups are respectively carried out in batches in the morning and afternoon every day for 30 d.
And in the process of each batch of 4h experiment, a xylene detector is used for monitoring in real time and maintaining the concentration of xylene gas in the box body in due time.
The audio signal of 4h in-process with the play of little audio amplifier preparation simultaneously, the model group animal produces the state of fear intermittently.
The small fan is used for generating air flow to improve the environment in the box body, and the rechargeable battery is used for timely providing power for the fan, the sound box and the detector.
Animals in the FC group (cage number FC1/FC2) listened to only the audio signal 4h per day.
And (5) processing animal sampling and detection after the model experiment is completed.
The experimental system of conditional fear (ZS-KJ, containing L abMaze animal behavioral analysis software and video and computer), xylene gas (Wuhan Nuede special gas Co., Ltd., 450ppm in 10L tank, 0.7MPa), animal gas toxicant exposure instrument (Beijing Huilong and technology Co., Ltd., HRH-CSED-K), pump-suction xylene detector (Zhengzhou Junda instrument Co., Ltd., JA908), mini-speaker (playing the audio file: 19min40s end in 20min per week, 75dB which starts to last for 20 s: 1000Hz until this cycle ends, 12 cycles, total time 4h), mini-fan (for air flow in toxicant exposure box to improve environment), charge pool (for supplying power for spare of detector, mini-speaker).
Measurement results of indicators of fear stress model:
in order to maintain the conditioned response fear state of the later-stage compound xylene inhalation model animals, further experiments on the fear memory regression time were carried out, the percentage of freezing time is shown in fig. 1, and the statistical results show that the percentage of freezing time of the animals in the fear model group F is still significantly higher than that in the control group C after 10W. The condition reaction fear memory generated only by the sound signal without electric stimulation can maintain at least 4W, and the fear state of the composite exposure experiment can be ensured. Through the record statistics of the weights of the grouped animals, the results show that the weight growth trends of the animals of the fear model group F and the animals of the control group C are not obviously different in the long-term experiment process.
The progress is good, and the behavior of the animal changes obviously along with the advancing of modeling time. The motion trail shows that the exploration behavior is reduced and the fear state is generated. Continuous monitoring of 6W freezing time shows that the fear model group F is significantly higher than the control group C, and the conditional fear animal model is successfully established.
Statistical results are shown in fig. 2 by recording the weights of the animals in the groups in the two experiments, and the results show that the weight growth trends of the animals in the fear model group F and the animals in the control group C are not significantly different in the long-term experiment process.
Referring to fig. 3, the movement traces of the model group and the blank control group showed a decrease in the exploratory behavior of the rats.
FIGS. 4-7 illustrate: fear stress can cause the obvious change of organism intestinal flora, and the species with the largest difference contribution degree are: firmicutes, Bacteroidetes and Proteobacteria.
As can be seen in FIG. 8, fear stress may cause abnormalities in lipid metabolism in the body, affecting the metabolic process of arachidonic acid.
The pathological examination result of the hippocampus is shown in FIG. 9. from FIG. 9, it can be seen that the FC group has some neuron retraction staining and neurofibrillary tangles are seen, and the F L group has some neuron retraction staining and neurofibrillary tangles are seen.
The pathological examination result of the cerebral cortex is shown in fig. 10, and it can be seen from fig. 10 that the integral structure of the cerebral cortex in group F is normal, the arrangement of neurons is disordered, a large amount of neuron retraction and deep staining can be seen, the nystagmus is unclear, no interstitial loose edema and capillary hyperemia dilation can be seen, no obvious inflammatory cell infiltration can be seen in the tissue, the arrangement of the neurons in group FC is neat, no interstitial loose edema and visible capillary mild hyperemia dilation can be seen, the arrangement of the neurons in group F L is disordered, a large amount of neuron retraction and deep staining can be seen, as shown in the figure, no interstitial loose edema and capillary hyperemia dilation can be seen, no obvious inflammatory cell infiltration can be seen in the tissue, the arrangement of the neurons in group C L is disordered, the number of neurons is small, and a small amount of neuron retraction.
The pathological examination result of the amygdala in the brain is shown in FIG. 11, and it can be seen from FIG. 11 that the glial cells in group C L are obviously proliferated.
The results of intestinal pathological examination are shown in FIG. 12, a small amount of intestinal villus epithelium exfoliation necrosis can be seen in FC, F L and FH groups, and a mild edema can be seen in submucosa.
The pathological examination result of liver is shown in FIG. 13, the gap between liver sinuses of group F L is obviously increased, and blood stasis is visible in the gap
The detection results of hypothalamus-pituitary-adrenal (HPA) axis related neurohormones CRH, ACTH and CORT in blood are shown in figures 14-16, which cause the increase of the contents of the hypothalamus-pituitary-adrenal (HPA) axis related neurohormones CRH, ACTH and CORT of rats, and indicate that the organism generates stress response; changes in the levels of DA, NE, 5-HT and ACH secretion in neurotransmitters may cause anxiety and insomnia, and the results are shown in FIGS. 17-20.
The method successfully establishes and evaluates the conditioned reaction fear stress animal model, completes a stable and repeatable molding method, and has no obvious body trauma to the model animal. The experimental results show that using the early modeling method, it is expected that the conditioned response fear memory generated by the acoustic signal alone can maintain at least 4W without electrical stimulation during the subsequent 4h/d exposure time course of each group. The process is good, and the fear molding is completed.
The animal model established by the invention can reflect the influence of human body caused by fear stress and harmful gas conditions in a closed environment, and provides a theoretical basis for improving the working environment of submarine crew in future by truly simulating the operation safety of submarine crew in the faced deep sea closed space, various training and the sudden conditions inside and outside the submarine, and the like.
It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for establishing a rat fear stress and gaseous pollutant compound exposure model is characterized by comprising the following steps:
(1) establishing a fear model: selecting 180-220g healthy male wistar rats, placing the rats in a closed environment, giving a 75dB 1000Hz sound signal, giving 1mA plantar current stimulation at the same time for 3s at the tail of the sound signal, repeating the cycle for 20 times every day, and normally feeding free edible water in the rest time; lasting for six weeks;
(2) establishing a fear stress and gaseous pollutant composite exposure model: sucking gaseous pollutants into the rat which is built by the fear model, wherein the sucking time is 4 hours every day and lasts for 30 days;
(3) and (5) sampling and detecting.
2. The method of claim 1 wherein said gaseous contaminant is xylene.
3. The method of claim 2, wherein the concentration of xylene inhaled by rats is 0.38 ppm.
4. The method of claim 2, wherein the concentration of xylene inhaled by rats is 3.8 ppm.
5. The method of claim 1, wherein said sample testing comprises general behavior, percent freezing time, central nervous system, immune system, and endocrine system.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106573916A (en) * | 2014-06-09 | 2017-04-19 | 武田药品工业株式会社 | Radiolabeled compounds |
| CN106691599A (en) * | 2017-02-22 | 2017-05-24 | 河南中医药大学 | Fear experiment stress device |
| CN110251266A (en) * | 2019-06-25 | 2019-09-20 | 中国人民解放军陆军军医大学 | Exposure contamination experiment device is combined in a kind of rat noise and carbon monoxide integration |
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- 2020-04-24 CN CN202010334604.5A patent/CN111436396A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106573916A (en) * | 2014-06-09 | 2017-04-19 | 武田药品工业株式会社 | Radiolabeled compounds |
| CN106691599A (en) * | 2017-02-22 | 2017-05-24 | 河南中医药大学 | Fear experiment stress device |
| CN110251266A (en) * | 2019-06-25 | 2019-09-20 | 中国人民解放军陆军军医大学 | Exposure contamination experiment device is combined in a kind of rat noise and carbon monoxide integration |
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