CN113412817A - IBS-D experimental animal model method for improving molding rate and reducing death rate - Google Patents
IBS-D experimental animal model method for improving molding rate and reducing death rate Download PDFInfo
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Abstract
The invention provides an IBS-D experimental animal model method for improving the molding rate and reducing the death rate, which selects adult mice as modeling experimental objects and comprises the following steps: step 1, colorectal dilatation: firstly, lightly touching the anus of the mouse to make the mouse completely discharge excrement; then, a tracheal cannula with an air bag is plugged into the rat anus, wherein the air bag end of the tracheal cannula is positioned in the rat anus, and the outer end of the tracheal cannula is fixed at the root of the rat tail; then the mouse is placed in a transparent observation chamber, and the transparent observation chamber can limit the mouse to move left and right; after the mouse calms, injecting gas into the air bag of the trachea cannula to ensure that the air bag has certain expansion pressure and stimulate the mouse colorectal; step 2, chronic restraint stress: the mouse was taken out of the transparent observation room, and the abdomen and four limbs of the mouse were fixed in the supine position. The invention can better and completely replicate the characteristics of diseases, and can reduce the death rate and improve the molding rate at the same time; meanwhile, the molding effect can be more durable and stable.
Description
Technical Field
The invention relates to the technical field of medical research, relates to an establishment method of an animal model, and particularly relates to an IBS-D (diarrhea-predominant irritable bowel syndrome) experimental animal model method for improving the molding rate and reducing the death rate.
Background
IBS (irritable bowel syndrome) is a common functional bowel disease, mainly shows repeated abdominal pain, and is accompanied with symptoms of relieving symptoms after defecation, frequent defecation change, abnormal stool character and the like. IBS has a high incidence rate, about 5-15% worldwide, the lowest prevalence in southeast Asia (7.0%), and the highest prevalence in south America (21.0%). Women have a higher prevalence than men, and are more susceptible to the disease in a long-term high-stress environment or childhood trauma. IBS is classified into diarrheal-type IBS (IBS-D), constipated-predominant IBS (IBS-C), mixed-type IBS (IBS-M) and atypical IBS (IBS-U) according to the Roman IV criteria. In China, IBS-D patients with diarrhea as the main symptom are more, and the IBS-D patients account for 66.3 percent of the incidence rate of the whole IBS. IBS-D is also a biopsychological and social disorder, which is accompanied by anxiety, depression and other mental disorders. IBS has complex symptoms, no specific medicine, high recurrence rate and low clinical treatment satisfaction, has great influence on physical and mental health of patients, causes excessive consumption of public medical resources, and becomes a research hotspot in the field of gastrointestinal diseases.
The establishment of a qualified animal model which is closer to clinical cases is the basis of disease research, so that continuous innovation and perfection of the IBS-D animal model have very important significance for animal experimental research.
The pathogenesis of IBS is complex and diverse, gastrointestinal motility and paresthesia are the pathophysiological basis, the pathogenesis of IBS is related to the change of the brain-intestinal axis function, and the IBS is a biological-psychological-social obstacle, so that the construction of an animal model has certain difficulty. The current modeling method can construct the visceral sensitivity and abnormal dynamic characteristics of IBS-D to a certain extent, but lacks the expression of emotional disorders such as anxiety, depression and the like, and cannot completely reproduce the characteristics of diseases. Meanwhile, a certain failure rate and death rate exist in the molding process.
In conclusion, most of the existing irritable bowel syndrome animal models are started from a certain aspect of pathophysiological mechanisms, and have limitations of different degrees, and the quality of the existing common model preparation method lacks accurate comparison research.
Disclosure of Invention
The invention provides an IBS-D experimental animal model method for improving the molding rate and reducing the death rate, which can better and completely replicate the characteristics of diseases, reduce the death rate and improve the molding rate; meanwhile, the molding effect can be more durable and stable.
In order to achieve the purpose, the invention provides the following technical scheme: an IBS-D experimental animal model method for improving the molding rate and reducing the death rate selects adult mice as a modeling experimental object, and comprises the following steps:
firstly, lightly touching the anus of the mouse to make the mouse completely discharge excrement; then, a tracheal cannula with an air bag is plugged into the rat anus, wherein the air bag end of the tracheal cannula is positioned in the rat anus, and the outer end of the tracheal cannula is fixed at the root of the rat tail; then the mouse is placed in a transparent observation chamber, and the transparent observation chamber can limit the mouse to move left and right; after the mouse calms, injecting gas into the air bag of the trachea cannula to ensure that the air bag has certain expansion pressure and stimulate the mouse colorectal;
Preferably, the specific process of injecting gas into the balloon of the endotracheal tube to stimulate the mouse colorectal in the step 1 is as follows: the balloon was allowed to expand at 60mmHg to 80mmHg to stimulate the mouse colorectal once a day for five minutes each time for two consecutive weeks of molding.
Preferably, the specific process of fixing the mouse in the supine position in the step 2 is as follows: the abdomen and the four limbs of the mouse are fixed by the magic fur-sticking surface, and the mouse is fixed on the binding frame in the supine position to limit the movement of the mouse; chronic restraint stress is stimulated once a day, two hours each time, and the model is continuously stimulated and molded for two weeks.
Preferably, the diameter of the tracheal cannula with the air bag is two to three millimeters; the trachea cannula is plugged into the anus of the rat by five to nine centimeters. Preferably, the diameter of the tracheal cannula is 2.7 mm, and the tracheal cannula is approximately 7cm plugged into the anus of the rat. The tracheal cannula with the diameter is beneficial to stimulating the mice, improving the molding rate of the mice and reducing the death rate.
Preferably, the transparent viewing chamber is twenty centimeters long, six centimeters wide and eight centimeters high. The transparent observation chamber with the size is beneficial to limiting the left and right movement of the rat, so that the rat can only move back and forth, and the phenomenon that the intubation tube damages the intestinal tract when the rat twists is prevented, and the death rate of model building is reduced.
Preferably, the binding frame is a thin plate, and the thin plate type binding frame has a length of twenty centimeters, a width of five centimeters and a thickness of five centimeters. The thin plate is beneficial to fixing the mouse and stimulating the mouse with chronic stress.
Preferably, the inflation pressure within the balloon is 80 mmHg.
Preferably, the adult mouse is a vista mouse, and the vista mouse is fifteen to twenty centimeters in length.
Compared with the prior art, the invention has the beneficial effects that:
1. because the etiology and pathogenesis of irritable bowel syndrome are complex, the characteristics of the disease cannot be completely reproduced although certain signs and symptoms can appear from a certain aspect. The invention adopts a molding method combining colorectal distension and chronic restraint stress, and utilizes compound physical mechanical stimulation to change the emotion and physiological state of animals, thereby completely leading a rat model to generate the characteristic of irritable bowel syndrome. Meanwhile, the intuitive model enables the mice to have obvious and intuitive symptoms and signs, is convenient for researchers to record, prepares a qualified animal model, and effectively reduces the death rate and improves the molding rate. Particularly, the stimulation is caused on the psychology aspect of the rat by combining the chronic constraint stress, the peripheral intestinal tract of the rat can be directly stimulated by colorectal dilatation, the central and peripheral co-stimulation mode can realize the synergistic interaction, the molding effect can be more durable and stable, the molding rate is improved, and the death rate caused by only adopting the colorectal dilatation method is also reduced. Compared with the single central stimulation model building by water avoidance, mother-infant separation and the like or the peripheral stimulation mode of direct drainage such as senna leaf lavage, acetic acid enema and the like, the combined stress model building method has stronger stimulation intensity of combined stress, and the colon non-inflammatory pathological changes are more in line with the characteristic pathological manifestations of IBS-D, so that the model mouse can perfectly simulate the disease characteristics.
2. The colorectal distension experiment has strong stability and repeatability, can be quantified, and can greatly avoid the animal from being harmfully stimulated by narcotics, additional operations and the like due to the sobering animal experiment which is not limited by activities, thereby obviously reducing the death rate of animals.
3. The invention overcomes the defect that the stimulation of chemical drugs (senna lavage, acetic acid enema and the like) in the prior art can cause the drugs to enter the systemic circulation so as to cause the change of the hormone level in the body. The invention adopts physical modeling, so that the model mouse has no drug circulation influence and has more objective indexes.
4. The invention adopts a constraint stress experiment after colorectal dilatation, wherein the constraint stress can cause a model mouse to generate psychological diseases, and in the process of causing psychosomatic diseases of the model mouse, the chronic stress can also stimulate the change of specific parts of the brain (mainly the change of a marginal system (especially hippocampus)) to cause the change of cognition and emotion, thereby conforming to the IBS-D brain-intestine interaction mechanism and improving the molding rate.
5. The colorectal distension is combined with the restraint stress modeling, the experimental animals have diarrhea caused by the physical stimulation of the colorectal, the change of intestinal motility and the change of emotion, and the combination of the diarrhea, the intestinal motility and the emotion can more comprehensively and truly simulate the intestinal function and the psychological change of IBS-D.
6. The modeling method of the invention is convenient for researchers to observe the general condition, weight change and diarrhea condition (loose stool rate) of each group of rats every day during the modeling period; and (4) stool water content, detecting abdominal wall withdrawal reflex (AWR) before and after molding, colon epithelium pathological indexes and the like to judge whether molding is successful. Therefore, the invention adds a stimulation factor causing IBS-D by a composite method to stimulate the physical and psychological stimulation, thereby simulating the onset of IBS-D to a greater extent; meanwhile, the molding method is relatively safe and noninvasive, so that the death rate of rats is greatly reduced.
Drawings
FIG. 1 is a graphical illustration of AWR scores;
FIG. 2 is a schematic diagram showing the weight gain of rats in each group before and after molding;
FIG. 3 is a schematic diagram showing moisture contents (%) of feces of rats in each group before and after molding;
FIG. 4 is a graph showing the comparison of AWR scores in groups of rats before modeling;
FIG. 5 is a graph showing the comparison of AWR scores in various groups of rats after molding;
FIG. 6 is a graph showing comparison of visceral pain threshold (mmHg) of rats in each group before and after molding;
FIG. 7 is a graph showing pathological results (HE,. times.200) of colonic epithelium in rats of each group;
FIG. 8 is a schematic representation of restraint stress;
fig. 9 is a schematic diagram of colorectal distension.
Detailed Description
Example 1:
adult male Wistar rats (Wistar rats) are selected as experimental objects, and an IBS-D rat model is established by adopting a colorectal distension and chronic constraint stress combined modeling method. The specific molding method is as follows:
colorectal dilation: before expansion, the anus of the rat is touched lightly to discharge the excrement, the minimum model 2.0 tracheal cannula with the air bag is used for smearing paraffin oil to lubricate the anus fully and then is filled into the anus for about 7cm, the position 1cm outside the anus is fixed at the tail root of the rat by a medical adhesive tape to prevent the rat from falling off, as shown in figure 9, the rat is placed in a transparent observation chamber (20 x 6 x 8cm) to limit the left and right movement of the rat, and only the rat can move back and forth; after it was completely calm, the colorectal was stimulated at a distending pressure of 80mmHg, 1 time per day for 5 minutes each time, and molding was continued for 2 weeks.
② chronic restraint stress: as shown in fig. 8, rats were fixed to a restraint frame (20 × 5cm) in a supine position, the abdomen and four limbs were fixed using a hook and loop fastener, and the activity was restricted by stimulating 1 time per day for 2 hours each time for 2 weeks.
In order to further illustrate the advantages of the IBS-D experimental animal model method for improving the molding rate and reducing the death rate, a comparison test is carried out on the IBS-D experimental animal model method for improving the molding rate and reducing the death rate.
1 content of the experiment
1.1 Experimental animals and raising environments
Selecting 40 male Wistar rats with SPF (specific pathogen free) grade, the weight of 160-200 g (the body length is 15-20 cm), purchasing from Youdoushuo laboratory animal company Limited, license number: SYXK (Chuan) 2020-. The feed is raised in an animal experiment center of Chinese medicine university of Chengdu, the temperature is controlled to be 22-24 ℃, the humidity is 40-60%, the day and night are 12 hours respectively, and the feed is noiseless and has no strong light. The food is free to drink water, and the feed is adaptively fed for 7 days. The experimental protocol has passed ethical examination of laboratory animals at the subsidiary hospital of Chinese medicine university of Chengdu (No. 2020 DL-001).
1.2 Experimental reagents and instruments
1.2.1 Experimental reagents
Hematoxylin staining solution, eosin staining solution (Shanghai Biyuntian biotechnology limited Co., Ltd., batch No.: C0105-1; C0105-2)
EG1150C Split Paraffin embedding machine, RM2235 Paraffin microtome (Leica, Germany)
DHG-9023A electric heating constant temperature air-blast drying oven (Shanghai extract macro experimental equipment Co., Ltd.)
Self-made colorectal dilator: the binding belt of the desk type mercury sphygmomanometer is removed, and the sphygmomanometer, the tracheal cannula (model 2.0) with the air bag and the pressurizing balloon are connected by using a three-way infusion pipe, so that the air tightness of each connecting part in water test and the stability of the mercury column after inflation are ensured. Self-made colorectal pressure measuring instrument: the trachea cannula of the self-made colorectal dilator is replaced by a disposable 8F balloon-free catheter, the tail end of the catheter is fixed with a 3-inch latex balloon with the length of 5cm by surgical silk threads and AB glue, and the pressurized balloon is replaced by a 20ml empty needle tube. Air leakage was tested in water by the same method as above.
Self-made binding frame: a rectangular block of 25X 5X 2cm was used as the main body, and 2 blocks of 5X 3cm were fixed to the lower end. The two sides of the wooden frame are fixed with magic tape stabbing surfaces (20 x 1.5cm), and the abdomen and four limbs of the rat are fixed by using magic tape hair surfaces.
1.3 animal modeling
40 rats were divided into a colorectal distension group, a chronic restraint stress group, a colorectal distension-combined chronic restraint stress group (combined group), a blank control group, and 10 rats each.
Colorectal dilation group: before expansion, the anus of a rat is touched lightly to discharge excrement, a 3.0 model air pipe with an air bag (the diameter of the air pipe is about 4 mm) is adopted, paraffin oil is smeared on the air pipe to be lubricated fully, the air pipe is plugged into the anus for about 7cm, the air pipe is fixed at the root part of the tail of the rat by a medical adhesive tape at the position 1cm outside the anus to prevent the air pipe from falling off, and then the rat is placed in a transparent observation chamber (20cm, 6cm and 8cm) to limit the left and right movement and only move back and forth; after the patient is completely calmed, injecting gas into the trachea of the air bag, stimulating the colorectal with the expansion pressure of 80mmHg for 2 times a day, lasting for 5 minutes each time, and continuously molding for 2 weeks.
Chronic restraint stress group: rats were fixed to a restraint frame (20cm x 5cm) in a supine position, and the abdomen and four limbs were fixed using a hook and loop fastener, and the activity was restricted by stimulating 1 time per day for 2 hours each time for 2 weeks.
Colorectal dilatation combined with chronic restraint stress group (combination group): the chronic restraint stress is carried out after the colorectal dilatation every day for two weeks by combining the colorectal dilatation method and the chronic restraint stress method. Namely: firstly, lightly touching the anus of a rat to discharge excrement, then adopting a 2.0 model trachea with an air bag (the diameter of the trachea is about 2.7 mm), smearing paraffin oil on the trachea to fully lubricate, then plugging the trachea into the anus for about 7cm, fixing the trachea at the root of the rat tail by a medical adhesive tape at a position 1cm outside the anus without dropping, and then placing the rat in a transparent observation chamber (20cm 6cm 8cm) to limit the left and right movement of the rat and only move back and forth; after it is completely calm, gas is injected into the trachea of the balloon, and the colorectal is stimulated at a dilating pressure of 80mmHg, 1 time per day, each time for 5 minutes. After colorectal distension was completed every day, rats were fixed on a restraint frame (20cm x 5cm) in a supine position, and the abdomen and four limbs were fixed using a hook and loop fastener, and the activity was restricted by stimulating 1 time a day for 2 hours each time. The combination group was stimulated for 2 weeks daily colorectal distension in combination with chronic restraint stress stimulation.
The rats in the blank control group were palpated for their perineum for 5 minutes 1 time per day for 2 weeks without additional stimulation.
1.4 specimen Collection
2% pentobarbital sodium is injected into abdominal cavity with 2.5ml/kg dosage for anesthesia, after rat anesthesia, the abdominal wall is cut open, the abdominal cavity is exposed, colon tissue with the end of 6cm is taken, the intestinal contents such as blood, feces and the like are washed away by normal saline, and fixed by 4% paraformaldehyde to prepare colon pathological section.
1.5 Observation index
1.5.1 general case
The mental state, the activity, the fur health cleanliness, the external stimulus reaction, the stool hardness and the particle number and the diet water intake of each group of rats in the experimental process are observed.
1.5.2 rat body weight
The rats are respectively weighed and recorded at the time points of 1 day before molding, one week after molding and after molding, so as to be convenient for counting and comparing the weight increase. During the modeling period of the IBS-D rats, the food intake is reduced, the weight is slowly increased, and even part of the weight is reduced.
1.5.3 conditions of diarrhea-water content of stool
Rat feces are collected by breeding rats in a single cage before and after molding, the collection time is 8: 00-12: 00Am, the rat feces are weighed (wet weight), and the rat feces are weighed (dry weight) after being dried by an oven. Calculating the formula: stool dry-wet specific gravity (%) - (wet-dry weight)/wet weight × 100%.
1.5.4 visceral sensitivity test-AWR and visceral pain threshold
The visceral sensitivity was measured in a home-made rat fixed observation room. The abdominal wall retraction reflex score detection was performed after molding, i.e., before and after treatment, for each rat under colorectal distension. The specific method comprises the following steps: before detection, a rat fasts for 12 hours without water prohibition, firstly lightly stroking the anus of the rat to ensure that residual excrement of the rat is discharged, under ether anesthesia (the anesthesia degree is based on the fact that the rat loses autonomous and persistent resistance), smearing the saccule with paraffin oil, then lightly putting the saccule into the colorectal of the rat to ensure that the top end of the saccule is 7cm away from the anus, fixing the saccule at the tail root of the rat by using a medical adhesive tape to prevent the saccule from falling off, and putting the rat into a fixer to prevent the rat from turning and moving left and right and only doing forward and backward movement. After the rats were then allowed to recover and acclimate, pressure was raised by continuous, slow insufflation, and responses to the intestinal lumen balloon dilatation stimulation were observed at four pressure gradients of 20, 40, 60, and 80mmHg, each pressure gradient was measured 3 times in succession for 20s at 5min intervals, and 3 AWR scores were averaged (see Table 1AWR score rules, FIG. 1AWR score schematic). The minimal pressure value corresponding to 3 point responses of rats observed with naked eyes, i.e. stronger contraction of abdominal wall and lifting off the plane of observation chamber, was used as the visceral pain threshold. A lower pain threshold indicates a higher tolerance of the rat to rectal balloon stimulation and vice versa. Visceral pain threshold was measured 3 times and averaged.
TABLE 1AWR score rules
1.5.5 colonic HE staining
1. After the materials are taken, a colon specimen is fixed by paraformaldehyde, and paraffin embedding is carried out after the fixation is finished.
2. The slices are dewaxed for 5-10 minutes by xylene conventionally, washed with various grades of ethanol to water: xylene (I)5min → xylene (II) 5min → 100% ethanol 2min → 95% ethanol 1min → 80% ethanol 1min → 75% ethanol 1min → distilled water washing 2 min.
3. Hematoxylin staining and washing away excess stain → distilled water washing → hydrochloric acid ethanol differentiation → warm water soaking
4. The solution was stained for 2min in eosin, followed by 2 washes with 70% ethanol.
5. Dehydrating with conventional ethanol and xylene, transparent, and sealing with neutral resin.
The gross lesion of the colon tissue is observed under a 40-fold electron microscope, and then an observation area is selected for 200-fold fine observation. The degree of inflammation was judged from the integrity of the colonic epithelium, ulceration, edema, vasodilation, inflammatory cell infiltration.
1.6 statistical analysis method
Each set of data was analyzed using SPSS23.0 software. Data of each group are calculated as mean ± sd
And (4) form representation. The One-way ANOVA test is adopted for the comparison among the groups, the LSD test is adopted for the uniform variance, and the Tamhane's T2 test is adopted for the irregular variance. If the data do not conform to normal distribution and the variances are not uniform, statistical analysis is carried out by adopting a K-W detection method in a non-parametric detection method rank sum test. The difference is statistically significant when P is less than 0.05.
2. Results of the experiment
2.1 mortality and Moldline
Mortality rate: in the experimental stage, 1 rat died in the colorectal distension group alone, and no rat died in the other groups.
The molding rate is as follows: combining with the symptom evaluation indexes (mental condition, weight change, diarrhea condition and viscera sensitivity evaluation) of the rat and laboratory examination (colon HE staining detection) to judge whether the molding is successful or not. Except that 1 rat pathology in the colorectal distension group indicated 'moderate inflammatory reaction', which is regarded as molding failure, was removed, and rats in the other model groups were successfully molded.
2.2 general conditions in rats
Blank control group: the rat has good spirit, smooth hair, color, whiteness, cleanness, normal eating and sensitive response to external stimulation.
Colorectal dilation group: the rat is poor in psychology, the autonomous activity is reduced, the rat is easy to stick or lean at the corner of the cage, and the resistance is weakened when the rat is grabbed; the drinking water amount is not reduced, the hair is slightly withered and yellow and scattered, and the color of the auricle is light.
Chronic restraint stress group: the mental changes of the rats are not obvious, the autonomic activity is reduced after being restrained on the day, and the activity amount is gradually recovered to be normal after the autonomic activity lasts for about 3 to 6 hours; the amount of dietary water intake was not reduced.
Combination group: the rat has reduced autonomous activity and poor spirit, and the ajar is crouched at the corner of the rat cage; the drinking amount of the diet is obviously reduced; the fur is disordered and has no color, the hair around the anus is visible and easy to be stained, and the hair is easy to fall off when being caught; the capture and binding counteractive reaction is weakened.
2.2 weight gain in rats
The weights of rats in each group before modeling are not obviously different, have no statistical significance (P is more than 0.05), are uniform at the base line and have comparability.
After one and two weeks of molding, compared with a blank control group, the weight increase difference of the rats in the chronic constraint stress group is not obvious, and the rats have no statistical significance (P is more than 0.05); the weight gains of the rats in the colorectal distension group and the rats in the combined group are obviously reduced, and the difference has statistical significance (P is less than 0.05), wherein the weight gains of the rats in the colorectal distension group are obviously less than those of the rats in the combined group, the weight gains are slow, and even part of the weights of the rats are increased negatively. Comprehensive literature reports that the IBS-D rats have reduced food intake, retarded weight increase and even partial weight reduction during modeling. Consistent with the results of this experiment. (see Table 2, FIG. 2)
TABLE 2 weight gain (g) for each group of rats
Note: p < 0.05 compared to control blank; compared with the colorectal expansion group,#p is less than 0.05; compared with the group of chronic restraint stress,▲P＜0.05
2.3 diarrhea-moisture content of stool
Before molding, the water content of the excrement of each group of rats has no obvious difference (P is more than 0.05); after the model is made, compared with a blank control group, the water content of the excrement of the rats in the chronic constraint stress group is not obviously changed (P is more than 0.05); the water content of the excrement of rats in the colorectal distension group and the combined group is obviously increased (P is less than 0.05). Compared with the chronic restraint stress group, the water content of the excrement of the rats in the combined group is obviously increased, and the difference is significant (P is less than 0.05). (see Table 3, FIG. 3)
TABLE 3 moisture content (%)
Note: p < 0.05 compared to control blank; compared with the colorectal expansion group,#p is less than 0.05; compared with the group of chronic restraint stress,▲P＜0.05
2.4 visceral sensitivity test-AWR vs visceral pain threshold
Before molding, when the pressure gradient is 20, 40, 60 and 80mmHg, the AWR scores of all groups are not different, the baseline is uniform and is comparable. (see Table 4, FIG. 4)
TABLE 4 comparison of AWR scores in groups of rats before modeling
After modeling, when the pressure gradient is 20 mmHg and 40mmHg, compared with a blank control group, the difference of the AWR scores of the colorectal distension group and the combined group is obvious and has statistical significance, the AWR score is increased and is more than 3 points, and the viscera shows high sensitivity. When the pressure increased to 60mmHg, the AWR score for the chronic restraint stress group was greater than 3 points and visceral sensitivity increased. When the pressure was increased to 80mmHg, there was no difference in the AWR scores of the groups. (see Table 5, FIG. 5)
TABLE 5 comparison of AWR scores in groups of rats after model creation
Note that: each group was stimulated by balloon dilatation of the same intensity, P < 0.05 compared to the blank control group
Before modeling, the visceral pain threshold of rats in each group has no obvious difference (P is more than 0.05); after modeling, compared with a blank control group, the visceral pain threshold of rats in the chronic constraint stress group is increased, but the difference is not obvious (P is more than 0.05); the visceral pain threshold of rats in the colorectal distension group and the combination group is obviously increased (P < 0.05). Compared with the chronic restraint stress group, the visceral pain threshold of the rats in the combined group is obviously improved, and the difference has statistical significance (P is less than 0.05). (see Table 6, FIG. 6)
TABLE 6 visceral pain threshold comparison (mmHg) for rats of each group before and after molding
Note: p < 0.05 compared to control blank; compared with the colorectal expansion group,#p is less than 0.05; compared with the group of chronic restraint stress,▲P＜0.05
2.5 pathological results
The colon tissues of the blank control group and the chronic constraint stress group have intact structures, continuous and complete intestinal mucosa epithelium, and orderly arranged cell structures, and have no situations of vasodilatation, interstitial edema, inflammatory cell infiltration and the like. The colon tissue structure of the combined group is intact, the intestinal mucosa epithelium is continuous, the cell structure is arranged orderly, and a small amount of inflammatory cell infiltration is occasionally seen. All the rats in the above groups meet the IBS-D pathological standard. The rats in the colorectal distension group basically accord with the pathological change of IBS-D, and 1 rat has discontinuous colon tissue intestinal epithelium, less and orderly cell arrangement and moderate inflammatory cell infiltration. (see FIG. 7)
3 summary of the invention
By comparing three modeling modes and making symptom evaluation, the modeling modes are respectively a mode of colorectal distension with single factor, chronic constraint stress and a mode of colorectal distension with two factors combined with chronic constraint stress stimulation, so as to establish an animal model with high modeling rate and low death rate and closer to the clinical characteristics of diarrhea-predominant irritable bowel syndrome.
Increased visceral sensitivity and gastrointestinal motility disorders are known to be the major pathophysiological basis for IBS-D. And is also an important criterion for evaluating the success of the IBS-D animal model. Mental status and weight changes were observed in each group of rats as behavioral assessments, diarrhea symptoms were judged using fecal water content, and visceral sensitivity was assessed using a 3 point pain threshold in combination with an AWR score. Results in comparison of different model groups, the combined group and the single-factor colorectal distension group have more obvious changes such as diarrhea symptoms, visceral sensibility increase and the like than the chronic restraint stress group, and the combined group is superior to the single-factor colorectal distension group. By comparing the mortality and the molding rate of three groups of model rats, it is found that: mortality rate: colorectal distension group > chronic restraint stress group-combination group; the molding rate is as follows: combination group > colorectal distension group > chronic restraint stress group; compared with a single factor, the combined modeling method is superior to the single factor, and the modeling by using the combined stimulation mode of colorectal distension and chronic restraint stress is more in line with the characteristic performance of IBS-D, and has the advantages of high modeling rate and low mortality.
The present invention should be considered as limited only by the preferred embodiments of the invention, and not limited to the above embodiments, and it should be understood that any modifications, equivalents and improvements made within the spirit and principle of the invention are included in the scope of the invention.
Claims (10)
1. An IBS-D experimental animal model method for improving the molding rate and reducing the death rate selects adult mice as a modeling experimental object, and is characterized by comprising the following steps:
step 1, colorectal dilatation:
firstly, lightly touching the anus of the mouse to make the mouse completely discharge excrement; then, a tracheal cannula with an air bag is plugged into the rat anus, wherein the air bag end of the tracheal cannula is positioned in the rat anus, and the outer end of the tracheal cannula is fixed at the root of the rat tail; then the mouse is placed in a transparent observation chamber, and the transparent observation chamber can limit the mouse to move left and right; after the mouse calms, injecting gas into the air bag of the trachea cannula to ensure that the air bag has certain expansion pressure and stimulate the mouse colorectal;
step 2, chronic restraint stress: the mouse was taken out of the transparent observation room, and the abdomen and four limbs of the mouse were fixed in the supine position.
2. The method of claim 1 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, comprising: the specific process of injecting gas into the air bag of the tracheal cannula to stimulate the mouse colorectal in the step 1 is as follows: the balloon was allowed to expand at 60mmHg to 80mmHg to stimulate the mouse colorectal once a day for five minutes each time for two consecutive weeks of molding.
3. The method of claim 1 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, comprising: the specific process of fixing the mouse in the supine position in the step 2 is as follows: the abdomen and the four limbs of the mouse are fixed by the magic fur-sticking surface, and the mouse is fixed on the binding frame in the supine position to limit the movement of the mouse; chronic restraint stress is stimulated once a day, two hours each time, and the model is continuously stimulated and molded for two weeks.
4. The method of claim 1 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, comprising: the diameter of the tracheal cannula with the air bag is two millimeters to three millimeters; the trachea cannula is plugged into the anus of the rat by five to nine centimeters.
5. The method of claim 1 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, comprising: the transparent observation chamber is twenty centimeters long, six centimeters wide and eight centimeters high.
6. The method of claim 3 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, which comprises: the binding frame is a thin plate, and the thin plate type binding frame is twenty centimeters long, five centimeters wide and five centimeters thick.
7. The method of claim 2 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, which comprises: the inflation pressure within the balloon was 80 mmHg.
8. The method of claim 1 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, comprising: the adult mice are selected from Wistar mice, and the body length of the Wistar mice is fifteen to twenty centimeters.
9. The method of claim 4 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, which comprises: the diameter of the tracheal cannula with the air bag is seven millimeters at two points.
10. The method of claim 4 for an experimental animal model of IBS-D with increased mortality from the standpoint of modeling, which comprises: the trachea cannula is plugged into the mouse anus by seven centimeters.
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