CN113143531B - Method for establishing pulmonary edema rabbit animal model - Google Patents
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- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
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- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
- G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
- G01N5/045—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder for determining moisture content
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Abstract
The invention discloses a method for establishing a rabbit animal model with pulmonary edema in the field of biological model construction, which aims to overcome the defect of generating complicated respiratory system symptoms by reducing heart rate or other external circulation influences in the prior art. Compared with the prior art of an oleic acid injection method, an epinephrine infusion method and a chloroform infusion method, in the technical scheme, the medicament is directly infused into the lung to change the concentration of lung water, so that mutagenesis is realized, the influence of heart rate or other external circulation is avoided, a mutagenesis model of complicating respiratory system symptoms is overcome, active substances on the surface of the lung are retained to the maximum extent (directly infused inside), and the influence of complications on experimental data is reduced.
Description
Technical Field
The invention belongs to the field of biological model construction, and particularly relates to a method for establishing a pulmonary edema rabbit animal model.
Background
At present, there are many methods for replicating experimental pulmonary edema/EVLW increasing animal models in China, wherein the most common methods are oleic acid injection method, epinephrine drip injection method and chloroform injection method, and research for establishing neurogenic pulmonary edema rabbit model by injecting fibrinogen, thrombin and the like through medulla oblongata pool is also available. In the pulmonary edema model established by the methods, pathological changes such as pulmonary congestion, pulmonary hemorrhage and the like are combined except the pathological change of pulmonary edema, and the pathological changes are represented as different ultrasonic signs under the lung ultrasound, so that the judgment of the lung water content by adopting the lung ultrasound is influenced.
In addition, the adrenaline injection will affect the cardiac function, which causes the increase of the heart rate and the oxygen consumption of the cardiac muscle, and is not beneficial to the observation of the influence of the simple pulmonary edema on the cardiac function. While oleic acid-based fatty acids, oleic acid infusion may also cause a decrease in alveolar surfactant in addition to pulmonary edema induction. Chloroform is a general anesthetic, which accelerates heart rate after infusion. And the clinical mechanism of the occurrence of the wet lung of the newborn is a series of respiratory symptoms caused by delayed or incomplete absorption of lung fluid after birth of the newborn.
Disclosure of Invention
In order to solve the above problems, it is an object of the present invention to provide a mutagenic model that facilitates circumventing heart rate or other external circulatory influences to overcome concurrent respiratory symptoms.
In order to achieve the purpose, the technical scheme of the invention is as follows: and injecting a medicament for changing the increase of the lung water content into the animal to be tested in an external connection and discontinuous way so as to increase the liquid content in the interstitial substance, the alveoli and the cells of the lung and further induce the model to generate the pulmonary edema lesion.
After the scheme is adopted, the following beneficial effects are realized: compared with the prior art of an oleic acid injection method, an epinephrine dripping method and a chloroform injection method, the technical scheme has the advantages that the medicament is directly injected into the lung to change the concentration of lung water, so that mutagenesis is realized, the heart rate or other external circulation influences are avoided, a mutagenesis model with complicated respiratory system symptoms is overcome, active substances on the surface of the lung (directly injected into the lung) are also retained to the greatest extent, and the influence of the complication on experimental data is reduced.
Further, the intermittent injection of the experimental animal comprises the following steps: 1) Dividing animals to be tested into a plurality of experimental groups;
2) Variables in the experimental group include the body position of the animal and the medicament injection mode;
3) Quantitative resuscitation was used during the infusion, and pulmonary ultrasound examination was performed after each resuscitation sac ventilation.
Has the advantages that: compared with the prior art for reducing complications, the technical scheme has the advantages that an intermittent perfusion mode is adopted, the bearing capacity of the lung is ensured, the internal circulation of the lung is constructed, the change of the lung after a certain amount of perfusion is observed at regular time, and the mutagenesis time period is found out and determined.
Further, in the step 3), the ultrasonic detection method is as follows:
i) Dividing each lung into a first area, a second area and a third area in sequence;
ii) each area is divided into upper and lower two parts, and then left, right, upper and lower sides are distinguished by a mark;
iii) The ultrasonic probe is perpendicular to the ribs in the ultrasonic scanning process, a supine position is adopted when a first region is scanned, a lateral position is adopted when a second region is scanned, and a prone position is adopted when a third region is scanned.
Has the beneficial effects that: 1. compared with the prior art for searching accurate mutagenesis time points, the technical scheme determines the ultrasonic imaging signs of the lung which are actually changed on the basis of the increase of the lung water content through the position change, and the method for injecting the physiological saline into the lung under the condition of alternating the left lateral lying position and the right lateral lying position is tried in consideration of the mobility of water under the action of gravity, and the physiological saline injected into the lung is more uniformly distributed in the lung through the position change.
2. Considering that the lower part of the lung after water injection is possibly seriously affected due to the action of gravity, in order to obtain an animal model with uniform and consistent lesion in the lung, a plurality of different water injection body positions are set for observation.
Further, in the step i), the area division method is that the area from the sternum lateral line to the axillary anterior line is a first area, the area from the axillary anterior line to the axillary posterior line is a second area, and the area from the axillary posterior line to the vertebrae lateral line is a third area.
Has the advantages that: the lesion location and the influence are determined by dividing the area.
Further, the method also comprises the step of obtaining lung tissues after the lesion is induced, wherein the step of obtaining the lung tissues comprises the following steps:
s1, for each group of animals to be tested, after the total injected medicament amount reaches 60-80 ml, killing the animals by adopting an air embolism method;
s2, cutting open the chest to take out the two lungs, ligating the roots of the pulmones on the two sides, and separating the trachea on a ligation line;
s3, wedge-shaped taking of lung tissues of a lesion part visible to naked eyes, and immediately placing the lung tissues in paraformaldehyde solution with the content of 3% -4%;
s4, placing the obtained tissue in a refrigerator for 24 hours at the temperature of 2-4 ℃, and taking out after the tissue is fixed;
s5, continuously slicing the tissue after paraffin embedding, wherein the thickness of the slices is 3-5 um.
Further, the paraffin sections were stained by hematoxylin-eosin staining method.
Further, the stained section is subjected to pathological analysis, and the analysis process is as follows:
a) Baking slices: placing the slices in a constant-temperature drying oven at 60 ℃ for 2h;
b) Conventional dewaxing hydration: placing slices in xylene for 5min and 2 times for dewaxing, and then respectively placing slices in 100%, 95% and 90% ethanol for 5min to remove xylene;
c) Washing with distilled water for 5min and 3 times, and washing off ethanol;
d) Staining with hematoxylin for 15min, and washing with running water for 15s;
e) Decolorizing with 1% hydrochloric acid alcohol for 3s, and washing with flowing water for 15min;
f) Eosin staining for 2min, washing with flowing water for 15s to remove floating color;
g) And (3) dehydrating: placing the slices in 90%, 95%, 100% ethanol for 5min, and xylene for 5min x 2 respectively to make the specimen transparent;
h) Taking out the transparent glass slide from the dimethylbenzene, dripping a proper amount of neutral gum, covering a cover glass to seal the specimen, and observing the pathological change of the lung tissue under an optical microscope.
Further, in the step S2, the dry lung weight is weighed, the dry lung weight weighing method includes the steps of completely absorbing the moisture on the surface of the lung by using absorbent paper, weighing the lung to obtain the wet lung weight, and then putting the lung tissue into a drying oven for 72 hours and weighing the lung tissue.
Furthermore, compared with the wet-dry ratio of normal lung tissues, the experimental animal with pulmonary edema lesion is determined.
Further, the medicament is normal saline.
Drawings
FIG. 1 is a graph showing the distribution of lung water in group A (70 ml of physiological saline was injected in the supine position) according to the example of the present invention;
FIG. 2 is the distribution of lung water in group B (70 ml of physiological saline is alternately injected in the supine and prone positions);
FIG. 3 shows the distribution of lung water in group C (70 ml of physiological saline was alternately injected into the left and right recumbent positions);
FIG. 4 is a general view of the lung after water injection into the lung via tracheal intubation;
FIG. 5 shows pathological changes of lung tissue after 70ml of physiological saline is injected into the lung of group A;
FIG. 6 shows the pathological changes of lung tissue after 70ml of physiological saline is injected into the lung of group B;
FIG. 7 shows the pathological changes of lung tissue after 70ml of physiological saline is injected into lung of group C.
Detailed Description
The following is further detailed by way of specific embodiments:
the establishment method of the pulmonary edema rabbit animal model is mainly characterized in that a medicament for changing the increase of the lung water content is injected into an animal to be tested in an external connection and discontinuous mode so as to increase the liquid content in lung interstitium, alveoli and cells and further induce the model to generate the pulmonary edema lesion. The detailed construction steps are as follows:
1 materials and methods
1.1 Experimental animals
6 male New Zealand rabbits are purchased from Beijing Keyu animal breeding center, and the animal quality license number is as follows: SCXK (Jing) 2017-0002. The weight is 2.8-3.1 kg, and the average weight is 2.9 plus or minus 0.1kg.
1.2 Main Experimental reagents and instruments
Pentobarbital sodium, 0.9% sodium chloride solution, heparin sodium injection, rabbit table, animal depilator, vascular forceps, scalpel, dissecting scissors, toothed forceps, non-toothed forceps, medical suture, no. 3.0 neonatal trachea cannula, resuscitation bag, 1ml, 5ml, 20ml syringe, scalp acupuncture, EDAN portable all-digital color ultrasonic diagnostic apparatus (Acclarix AX 8), linear array probe (model: L10-4Q), frequency 9-12MHz.
1.3 animal ethical applications
The experiment was reviewed by the welfare ethics committee of laboratory animals at the fifth medical center of general hospital of the liberation force.
1.4 Experimental methods
1.4.1 animal groups
The animals were divided into 3 groups of 2 animals each. Injecting 0.9% sodium chloride into the trachea cannula of the animals in the supine position in the group A; the animals in group B adopt an alternate method of supine position and prone position to carry out gradient injection of 0.9% sodium chloride in the tracheal intubation; group C animals underwent endotracheal intubation with 0.9% sodium chloride gradient infusion using the left and right lateral decubitus alternating method.
1.4.2 animal preparation and anesthesia
The animals are fasted for 12 hours, weighed and placed in a head fixing box, the ear vein is plucked, iodophor is used for disinfection, and 20% pentobarbital sodium and 20-40mg/Kg are injected into a 5ml syringe through the ear vein.
1.4.3 depilation
After the anesthesia is successful, the rabbit is taken out of the fixing box, and the front chest, the back and the root parts and the neck parts of the two upper limbs of the rabbit are depilated by an animal depilator.
1.4.4 endotracheal tube
Fixing a rabbit in a supine position on an anchoring table, fixing limbs and a head, disinfecting with iodophor, paving a towel, making an incision about 2-3cm long in the center of a neck, cutting skin, separating subcutaneous tissues, muscles and fascia bluntly by using forceps, finding out a trachea, transversely cutting the front wall of the trachea at the 3 rd cartilaginous ring below a cricoid cartilage, making a longitudinal incision about 0.5cm long upwards to make the whole incision be in a T shape, cleaning local exudation, placing a No. 4 operation line below the trachea, placing a No. 3.0 trachea cannula with an air bag in the trachea through the incision, inserting the trachea cannula by the depth of 2cm, fixing the trachea cannula on a 21 trachea by using the operation line, observing the fluctuation of the thoracic cage on two sides after positive pressure ventilation by a resuscitation bag, if the fluctuation of the thoracic cage is good and the respiratory sounds on two sides are symmetrical, and seeing autonomous respiration after the resuscitation bag is taken off, thereby indicating that the intubation is successful.
1.4.5 preparation of the model
Injecting 10ml of normal saline into the group A animals in a supine position through a tracheal cannula, carrying out lung ultrasonic examination after 3-5 min of resuscitation bag positive pressure ventilation, continuously injecting 10ml of normal saline, carrying out lung ultrasonic examination after 3-5 min of resuscitation bag positive pressure ventilation until the accumulated water injection amount reaches 70ml, and recording the time from the 1 st water injection to the completion of ultrasonic examination after the last 1 water injection.
And B, injecting physiological saline into the trachea cannula by adopting a supine position and prone position alternating method, namely injecting 5ml of physiological saline into the supine position during each water injection, ventilating the resuscitation bag at positive pressure for 1-2 min, changing the prone position, injecting 5ml of physiological saline again, ventilating the resuscitation bag at positive pressure for 1-2 min, and then carrying out lung ultrasonic examination until the accumulated water injection amount reaches 70ml.
And C, injecting physiological saline into the trachea cannula by adopting a left lateral decubitus and right lateral decubitus alternating method, namely injecting 5ml of physiological saline into the left lateral decubitus during each water injection, ventilating the resuscitation bag at positive pressure for 1-2 min, changing the right lateral decubitus, injecting 5ml of physiological saline again, ventilating the resuscitation bag at positive pressure for 1-2 min, and then carrying out lung ultrasonic examination until the accumulated water injection amount reaches 70ml.
1.4.6 Lung ultrasound examination
Each lung is divided into 3 regions, the region from the parasternal line to the anterior axillary line is a first region (the left lung is marked as the L first region, and the right lung is marked as the R first region), the region from the anterior axillary line to the posterior axillary line is a second region (the left lung is marked as the L second region, and the right lung is marked as the R second region), the region from the posterior axillary line to the paraspinal line is a third region, and the two lungs have 6 regions (the left lung is marked as the L3 region, and the right lung is marked as the R third region). Each zone is divided into an upper part and a lower part, wherein the upper part of the first zone L is marked as L1/1, the lower part of the first zone L is marked as L1/2, and the like. And respectively carrying out ultrasonic scanning on each region of the bilateral lungs, wherein an ultrasonic probe is vertical to the ribs, the supine position is adopted when the first region is scanned, the lateral position is adopted when the second region is scanned, and the prone position is adopted when the third region is scanned.
1.4.7 pathological analysis of Lung tissue after physiological saline is injected into Lung through tracheal cannula
1.4.7.1 obtaining Lung tissue
To every group rabbit, after the volume of injected physiological saline reaches 70ml cumulatively, adopt the air embolism method to sacrifice the animal, open the chest fast and take out two lungs, ligate at two sides lung door root, prevent that the lung water from overflowing, after leaving the severed trachea on the ligature line, the lung tissue wedge of the visual pathological change position of naked eye is placed immediately in 4% paraformaldehyde in refrigerator 4 degrees centigrade after drawing materials (3mm. 3mm) and is preserved 24 hours and take out after fixing, the continuous section after the paraffin embedding, the piece thickness is 5um.
1.4.7.2HE staining
Hematoxylin-eosin staining (HE staining) is one of the most commonly used staining methods for morphological observation as one of the staining methods commonly used in paraffin sectioning technology. The hematoxylin staining solution is alkaline, and mainly causes chromatin in cell nuclei and ribosome in cytoplasm to be colored into purplish blue; eosin is an acidic stain that primarily reddens components in the cytoplasm and extracellular matrix.
1.4.7.3 pathological analysis procedure
a) Baking slices: placing the slices in a constant-temperature drying oven at 60 ℃ for 2h;
b) Conventional dewaxing hydration: placing the slices in xylene for 5 min-2 times for dewaxing, and respectively placing the slices in 100%, 95% and 90% ethanol for 5min to remove the xylene;
c) Washing with distilled water for 5min and 3 times, and washing off ethanol;
d) Staining with hematoxylin for 15min, and washing with flowing water for 15s;
e) Decolorizing with 1% hydrochloric acid alcohol for 3s, and washing with flowing water for 15min;
f) Eosin staining for 2min, washing with flowing water for 15s to remove floating color;
g) And (3) dehydrating: placing the slices in 90%, 95%, 100% ethanol for 5min, and xylene for 5min x 2 respectively to make the specimen transparent;
h) And taking out the transparent glass slide from the dimethylbenzene, dripping a proper amount of neutral gum, covering a cover glass and sealing the specimen. The pathological changes of the lung tissue were observed under an optical microscope.
1.4.8 Rabbit Lung Wet and Dry weight measurement
After the experiment, the animal is killed by adopting an air embolism method, the chest is opened immediately to dissect the lung from the heart and the great vessel, the trachea is separated at the carina part, the trachea is closed by a vascular clamp, then the two lungs are taken out, ligation is respectively carried out at the lung portals at the two sides, the main bronchus is separated at the proximal end after ligation, and the liquid in the lung is prevented from flowing out, as shown in figure 2. The water on the lung surface is sucked and cleaned by absorbent paper, wedge-shaped material (3 mm x 3 mm) of a lesion part visible in the flesh eye is taken, the wedge-shaped material is immediately placed in 4% paraformaldehyde for storage at 4 ℃, and the rest lung tissue is weighed and recorded as the lung wet weight. The lung tissue was then placed in a drying cabinet (80 degrees) for 72 hours and weighed, i.e., the dry lung weight.
2 results
2.1 duration of experiment
The average time of the experiment in the group A is 66min, the average time of the experiment in the group B is 151min, and the average time of the experiment in the group C is 58min.
2.2 Lung ultrasound scanning results
Referring to fig. 1, as the amount of water injected increases, line a disappears from each segment of the lung, the pleural line blurs, breaks, and disappears, and the number of line B gradually increases, gradually becoming a fused line B and a dense line B from line B, with the occurrence of lung consolidation under the pleural space.
The group A adopts supine position water injection, the lung water is not uniformly distributed, when the water injection amount is accumulated to the maximum amount of 70ml, the first area of the two lungs still has no obvious B line, the pleural line of the area 2 and the third area is fuzzy and disappears, a large amount of B lines appear, and the lung consolidation under the pleural can be seen.
Referring to fig. 2, in the first step, the pleural line of the first region of the left lung is smooth, regular and continuous, and part of the line a disappears, and the line B is visible; in the second area of the left lung, the pleural line is fuzzy, the line A disappears, and the line B and the lung excess change can be seen; thirdly, the pleural line in the third area of the left lung is blurred and disappears, the line A disappears, and the lung excess change can be seen; next, no obvious lesions were seen in the first region of the right lung; second, the pleural line of the second region of the right lung is fuzzy and discontinuous, and a large amount of B lines and a small range of lung excess change can be seen; in the third area of the right lung, the pleural line disappears, the line A disappears, and the lung consolidation becomes visible.
The group B adopts alternate water injection in the supine position and the prone position, when the water injection amount is accumulated to 70ml, the line B can be seen in the first area of the double lungs, but the pleural line can be seen to be interrupted, discontinuous or even disappear in the area 2 and the third area, and the lung consolidation under the pleura can be seen. As shown in fig. 4, in the first area of the left lung, the pleural line is clear, line a disappears, and line B is visible; in the second area of the left lung, the local pleural line disappears, the line A disappears, and a large amount of lines B and local lung excess change are visible; in the third area of the left lung, the pleural line disappears, the line A disappears, and a large number of lines B are visible; the second area of the right lung, the pleural line is smooth, clear and regular, the line A disappears, and the line B is visible; in the second area of the right lung, the pleural line is fuzzy and discontinuous, line A disappears, and line B and lung excess change are visible; in the third area of the right lung, the pleural line disappeared, line A disappeared, and line B and lung excess became visible.
The group C adopts the left lateral clinostatism and the right lateral clinostatism for alternate water injection, and when the water injection amount reaches 70ml, the first area, the second area and the third area of the double lungs can all see the symptoms of lung water increase with more consistent pathological changes. As shown in FIG. 3, the first region, 2 regions and 3 regions of the left lung and 1 region, 2 regions and 3 regions of the right lung are sequentially arranged from top to bottom, the pathological changes of the regions are basically consistent, the pleural line is thickened, the line A disappears, and a large amount of lines B can be seen.
2.3 anatomical general appearance of Lung after intrapulmonary Water infusion
After 70ml of water is injected in a cumulative way, the dissected two lungs are generally observed, the appearance of the two lungs is enlarged, the envelope is tense, and the normal lung tissue and the abnormal lung tissue are changed in a red-white alternative mode, as shown in figure 4.
2.4 pathological changes of lung tissue after intrapulmonary water injection through tracheal intubation
The alveolar structure composed of extremely thin flat epithelial cells can be seen in the tissue, and the alveolar sacs fused by a plurality of alveoli can be seen locally; the alveolus is separated by a thin highly vascularized collagen fiber and elastic fiber layer which forms the alveolus with the flat epithelium on the surface layer of the two alveolus; in the group B lung tissues, neutrophil disseminated infiltration can be seen in part of alveolar cavities, part of alveolar epithelial cells grow, and alveolar walls thicken, as shown in FIG. 6.
In fig. 5, after 70ml of physiological saline is injected into the lung of the left group A, the pathological change of the lung tissue is caused, the alveolar structure consisting of extremely thin flat epithelial cells is visible in the tissue, and the alveolar sac formed by fusing a plurality of alveoli is partially visible; the alveoli are separated by a thin layer of highly vascularized collagen fibers and elastic fibers that make up the alveolar septum with the flattened epithelium lining the two alveoli.
In FIG. 6, after 70ml of physiological saline is injected into the lung of group B, the pathological changes of lung tissues are caused, the alveolar structure consisting of extremely thin squamous epithelial cells is visible in the tissues, and the alveolar sacs fused by a plurality of alveoli are locally visible (rightmost arrows); in some alveolar spaces, there is a diffuse infiltration of neutrophils (arrow in the middle), and in some alveolar epithelial cells, there is a proliferation and thickening of alveolar walls (arrow on the far left).
Referring to fig. 7, after 70ml of physiological saline is injected into the lung of group c, the pathological changes of the lung tissue are observed, the alveolar structure composed of extremely thin squamous epithelial cells is observed in the tissue, and the alveolar sacs fused by a plurality of alveoli are locally observed; the alveolar spaces are separated by a thin, highly vascularized layer of collagen fibers and elastic fibers that make up the alveolar spaces with the flattened epithelium lining the two alveoli.
2.5 Lung Wet-to-Dry ratio
After the experiment is finished, the animal is killed by adopting an air embolism method, the chest is opened quickly, the two lungs are taken out, ligation is carried out on the root parts of the lung portals on the two sides, the lung water is prevented from overflowing, the trachea is separated on a ligation line, and the lung surface water is completely sucked by using absorbent paper and then weighed, namely the lung wet weight is obtained. The lung tissue was then placed in a dry box (80 degrees) for 72 hours and weighed, i.e., the dry lung weight, and the wet-to-dry lung ratio was calculated, with the results shown in table 1.
TABLE 1 Rabbit body weight and Lung moisture to dryness ratio
| Grouping | Rabbit weight (Kg) | Wet Lung weight (g) | Dry lung weight (g) | Dry-wet ratio of lung |
| A1 | 2.9 | 32.35 | 2.58 | 12.54 |
| A2 | 2.8 | 31.60 | 2.31 | 13.68 |
| B1 | 3.1 | 42.60 | 2.74 | 15.55 |
| B2 | 2.9 | 37.80 | 2.45 | 15.43 |
| C1 | 3.0 | 36.00 | 2.62 | 13.74 |
| C2 | 2.9 | 35.15 | 2.34 | 15.02 |
3 conclusion
Since we wanted an animal model of increased lung water content, the saline in the infused lungs was not aspirated. The literature indicates that the lung edema is considered to exist when the wet-to-dry ratio of normal lung tissue is less than 5 and more than or equal to 5. The lung dryness-wetness weight result of the experiment shows that the lung dryness-wetness ratio is obviously increased after water injection, which indicates that the lung water content is obviously increased. In addition, some researchers move the animal from a supine position to a prone position in order to achieve a uniform degree of damage throughout the lung. We also used the same experimental method for reference, and in order to obtain an animal model with more consistent lesion degree in the lung, the posture of the rabbit is changed in the process of injecting water into the lung. However, in the feasibility study of establishing the EVLW increasing rabbit model by injecting physiological saline into the lung through the tracheal cannula, the rabbit EVLW increasing model with relatively uniform lung lesions is not obtained by the method of alternately injecting the physiological saline into the supine position and the prone position, and the experimental time is obviously prolonged due to the change of the body positions, the lung has real ultrasonic imaging signs on the basis of the increase of the lung water content, and an ideal pure EVLW increasing rabbit model is not obtained. Considering the mobility of water under the action of gravity, a method for injecting physiological saline into the lung under the condition that a left lateral lying position and a right lateral lying position are alternated is tried, the physiological saline injected into the lung is uniformly distributed in the lung by changing the body position, and the lung ultrasound proves that the lung water increase rabbit model with uniform lung lesion is obtained by the method.
After the 3 groups of rabbits adopt different body positions and inject physiological saline into the lungs through the tracheal cannula, the lung ultrasonic scanning obtains the lung ultrasonic signs of line A reduction and line B increase, and the line B increase is the characteristic expression of the lung ultrasonic increasing by EVLW. The lung is obviously swollen after physiological saline is injected into the lung through the tracheal cannula, and the wet-dry ratio of lung tissues is obviously increased after water injection through the measurement of the wet-dry ratio of the lung, which is the gold standard for evaluating the increase of EVLW content. Both of these cases demonstrate that the method of transtracheal intrapulmonary infusion of saline can establish an animal model of increased EVLW.
Considering that the lower part of the lung after water injection is possibly seriously affected due to the action of gravity, in order to obtain an animal model with uniform and consistent lesion in the lung, three different water injection body positions are set for observation. The group A is water injection in a whole supine position, and the lung ultrasonic results show that the lung pathological changes in different areas are different, the pathological change in the third area is obviously heavier than that in the first area and the area 2, and the third area has obvious lung consolidation symptoms except the increase of lung water, and the condition that the water filling time in the alveolus of lung tissue in the third area is longer, so that the inactivation of lung surfactant in the alveolus causes the lung ultrasonic change similar to the secondary respiratory distress syndrome.
In the group B, water is injected by adopting alternate positions of the supine position and the prone position, areas 1, 2 and 3 have obvious lesions, but the experiment time is obviously prolonged due to the change of the positions, the lung ultrasonic scanning also has obvious lung consolidation symptoms, and an ideal rabbit model with increased pure lung water content is not obtained.
The group C animals adopt a method of alternating left lateral clinostatism and right lateral clinostatism to inject physiological saline into the lung, ultrasonic scanning of the lung is found, the lines A in the region 1, the region 2 and the region 3 disappear, a large number of lines B appear, the pathological changes of the three regions are uniform, no obvious lung excess symptom is seen, the experimental time and the time of the group A are not obviously prolonged, and the rabbit model with the lung water content increase and the pathological changes in the lung are uniform is obtained.
By combining the analysis, a rabbit model with increased lung water content can be established by directly injecting physiological saline into the lungs through a tracheal cannula, and the lung ultrasonic results obtained in three water injection positions are compared, so that the rabbit model with increased lung water content with uniform and consistent lung lesions can be obtained by injecting the physiological saline into the lungs by adopting a method of alternating a left lateral decubitus position and a right lateral decubitus position.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
The foregoing is merely an example of the present invention, and common general knowledge in the field of known specific structures and characteristics of the embodiments has not been described in detail, so that a person of ordinary skill in the art can understand all the common technical knowledge in the field of the invention before the application date or the priority date, can know all the prior art in the field, and have the ability to apply routine experimentation before the application date. It should be noted that, for those skilled in the art, without departing from the structure of the present invention, several changes and modifications can be made, which should also be considered as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be defined by the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (8)
1. The method for establishing the pulmonary edema rabbit animal model is characterized by comprising the following steps of: injecting a medicament for changing the increase of the lung water content into the animal to be tested in an external connection and discontinuous way so as to increase the liquid content in the interstitial substance, the alveoli and the cells of the lung and induce the model to generate the pulmonary edema lesion;
the intermittent injection of the experimental animal comprises the following steps: 1) Dividing animals to be tested into a plurality of experimental groups;
2) Variables in the experimental group include the body position of the animal and the medicament injection mode;
3) Quantitative resuscitation is utilized in the injection process, and ultrasonic lung examination is carried out after each resuscitation bag is ventilated;
in the step 3), the ultrasonic detection method comprises the following steps:
i) Dividing each lung into a first area, a second area and a third area in sequence;
ii) each area is divided into upper and lower two parts, and then left, right, upper and lower sides are distinguished by a mark;
iii) The ultrasonic probe is perpendicular to the ribs in the ultrasonic scanning process, a supine position is adopted when a first region is scanned, a lateral position is adopted when a second region is scanned, and a prone position is adopted when a third region is scanned.
2. The method of establishing a pulmonary edema rabbit animal model according to claim 1, wherein: the area division method in the step i) is that the area from the sternum line to the anterior axillary line is a first area, the area from the anterior axillary line to the posterior axillary line is a second area, and the area from the posterior axillary line to the vertebra line is a third area.
3. The method of establishing a pulmonary edema rabbit animal model according to claim 1, wherein: the method also comprises the step of obtaining lung tissues after the lesion induction, wherein the step of obtaining the lung tissues comprises the following steps:
s1, for each group of animals to be tested, after the total injected medicament amount reaches 60-80 ml, killing the animals by adopting an air embolism method;
s2, cutting open the chest to take out the two lungs, ligating the bottoms of the hilum of the two sides, and separating the trachea from the ligation line;
s3, wedge-shaped taking of lung tissues of lesion parts visible to naked eyes, and immediately placing the lung tissues in a paraformaldehyde solution with the content of 3-4%;
s4, placing the obtained tissue in a refrigerator for 24 hours at the temperature of 2-4 ℃, and taking out after the tissue is fixed;
and S5, continuously slicing the tissue after paraffin embedding, wherein the thickness of the slice is 3-5 um.
4. The method of establishing a pulmonary edema rabbit animal model according to claim 3, wherein: the paraffin sections were stained by hematoxylin-eosin staining.
5. The method of establishing a pulmonary edema rabbit animal model according to claim 4, wherein: and (3) carrying out pathological analysis on the stained section, wherein the analysis process is as follows:
a) Baking slices: placing the slices in a constant-temperature drying oven at 60 ℃ for 2h;
b) Conventional dewaxing hydration: placing slices in xylene for 5min and 2 times for dewaxing, and then respectively placing slices in 100%, 95% and 90% ethanol for 5min to remove xylene;
c) Washing with distilled water for 5min 3 times, and washing with ethanol;
d) Staining with hematoxylin for 15min, and washing with flowing water for 15s;
e) Decolorizing with 1% hydrochloric acid alcohol for 3s, and washing with flowing water for 15min;
f) Eosin staining for 2min, washing with flowing water for 15s to remove floating color;
g) And (3) dehydrating: placing the slices in 90%, 95%, 100% and 100% ethanol for 5min, and xylene for 5min × 2 respectively to make the specimen transparent;
h) Taking out the transparent glass slide from the dimethylbenzene, dripping a proper amount of neutral gum, covering a cover glass to seal the specimen, and observing the pathological change of the lung tissue under an optical microscope.
6. The method of establishing a pulmonary edema rabbit animal model according to claim 3, wherein: and in the step S2, weighing the dry lung weight, wherein the dry lung weight is weighed after completely absorbing the moisture on the surface of the lung by using absorbent paper, namely the wet lung weight, and then weighing the lung tissue after putting the lung tissue into a drying box for 72 hours.
7. The method of establishing a pulmonary edema rabbit animal model according to claim 6, wherein: and (4) determining the experimental animal with the pulmonary edema lesion by controlling the wet-dry ratio of the normal lung tissue.
8. The method of establishing a pulmonary edema rabbit animal model according to any one of claims 1 or 3, wherein: the medicament is physiological saline.
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