CN116712205A

CN116712205A - Tool set for preparing acute lung injury mouse model

Info

Publication number: CN116712205A
Application number: CN202310728170.0A
Authority: CN
Inventors: 赵帅; 李卉; 刘涛; 戴茹萍; 罗伟
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2023-06-20
Filing date: 2023-06-20
Publication date: 2023-09-08

Abstract

The invention provides a tool set for preparing an acute lung injury mouse model, which comprises an injector, two draw hooks, a suture needle, a suture line, one or more scissors and a tracheal cannula sleeve needle consisting of a medical disposable blunt needle and an outer sleeve of a human radial artery puncture needle matched with the medical disposable blunt needle. The invention also provides application of the tool kit in the process of preparing an acute lung injury mouse model, and the application process comprises the following steps: anesthesia, neck shaving, mouse fixation, skin incision and tracheal intubation, extraction of a guide core, injection of lung injury liquid, kneading and pressing of mouse chest and neck suturing, and thermal insulation resuscitation. The tool set can be used for preparing the acute lung injury mouse model efficiently, accurately, safely and cost-effectively, and is considered to have large-scale popularization and application values.

Description

Tool set for preparing acute lung injury mouse model

Technical Field

The invention belongs to the field of medical tools, and particularly relates to a tool set for preparing an acute lung injury mouse model.

Background

In the experimental mammal, the mice are small in size, convenient to raise and manage, easy to control, quick in production and propagation, and the most deeply studied, have definite quality control standards, and already have a large number of inbred lines, mutant lines and closed groups, so that the use amount is the largest in various experimental researches, and the use is the largest. ARDS, acute respiratory distress syndrome, ALI, acute lung injury, ARDS/ALI are clinically common critical conditions, and are the leading factors leading to death of patients. In the study of the mechanism of lung injury and drug development, a large number of acute lung injury mouse models are required. Therefore, how to efficiently prepare a mouse model of acute lung injury in batches becomes a problem that must be solved in the art.

The existing preparation method of the acute lung injury mouse model comprises a traditional nasal drip method, an intra-airway injection method, a surgical trachea cannula method, a tracheal blindness insertion administration method and the like.

The traditional nasal drip method has the advantages of no wound, but has the defects that the success rate of the nasal drip induced acute lung injury model and the stability of experimental results are interfered by various factors; the long distance from nasal cavity to lung can lead to loss or retention of part of the lung lesion, such as endotoxin LPS, a process that is strongly linked to individual variability in mice, and thus differences in the amount of lung lesion eventually flowing into the lungs will lead to model-induced instability.

The intra-airway injection method belongs to invasive operation, and has the defects that: 1. the small airway of the mouse can cause penetration of the whole airway layer in the process of injector puncture, and finally, the injection failure or leakage of lung injury liquid can be caused; 2. intra-airway injection is difficult to visually penetrate to the near-pulmonary end below the cricoid; 3. the intra-airway injection is difficult to perform in an upright position, and lung injury liquid is easily expectorated from the airway, ultimately resulting in model-induced instability.

Surgical endotracheal intubation requires incision of the mouse's trachea, and thus results in reduced survival of experimental animals. This method has long been eliminated because it is prone to direct death in mice.

Tracheal blindness dosing is published by 2023, 3, 31: noninvasive Intratracheal Lipopolysaccharide Instillation in Mice has recently been proposed, which has the advantage of being non-invasive. Specifically, fig. 2 therein shows a cannula tool, i.e. a cannula kit and its components; comprises a pen lamp, an optical fiber and a sleeve. Also written herein is: 20-30g mice can use 22g catheters (2.5-3.8 cm long); the cannula is assembled to the cannula pen and the light of the cannula pen is turned on. Wherein air was pumped into the airway of the mice using a pasteur pipette; during modeling, if a cannula is successfully inserted into the trachea of a mouse, the chest of the mouse will swell; if the cannula is inserted into the esophagus of the mouse by mistake, the right lower abdomen of the mouse is swelled; thus, the method uses a method of pumping air with a Pasteur pipette to determine if the cannula was successful. According to the description, whether the intubation is successful or not can be judged theoretically through the fluctuation of the abdominal cavity and the thoracic cavity of the mouse; however, in practice, even with a rich intubation experience, the anesthesiologist may misinterpret the esophagus as an airway during a clinical intubation procedure, resulting in a failure of the tracheal intubation, and thus the clinical gold standard for judging whether a patient is successfully intubated is to use end-of-breath CO ₂ Detecting to identify the position of the cannula; if the intubation tube is mistakenly inserted into the esophagus, the intubation tube is immediately changed to ensure the life safety of the patient. The mice have small sizes, cannot be judged by the method, and are difficult to be inserted. That is, the cannula error rate and the judgment error rate of the modeling method in this document are very high, and particularly, the difficulty is quite large for a novice operator. The document also describes that a great deal of practice is required to master these technical skills; in addition, for mice with smaller sizes, the intubation tube is difficult to insert into the trachea, and the trachea is easy to scratch in the operation process; therefore, it is suggested in this document to select mice of larger body size for the experiment. Therefore, the tracheal blind insertion drug administration method has low modeling success rate due to low accuracy, and the modeling staff has slow hands when operating; the tracheal tube is easy to enter the esophagus, so that even a skilled operator has a larger probabilityFailure of the rate.

Thus, there is a need in the art for a new method of preparing a mouse model of acute lung injury and for a new modeling tool kit.

Disclosure of Invention

Accordingly, the present invention first provides a kit for preparing a mouse model of acute lung injury, the kit comprising a syringe, two hooks, a suture needle, one or more scissors, and a tracheal cannula sleeve comprising a medical disposable blunt needle and an outer cannula of a human radial artery puncture needle matched with the blunt needle.

In the invention, the outer sleeve of the human radial artery puncture needle is also called an arterial puncture catheter or simply called a catheter; the medical disposable blunt needle is also called a cannula guide core, or simply a guide core. The medical disposable blunt needle is inserted into the radial inner side of the outer sleeve of the human radial artery puncture needle from the upper end of the outer sleeve matched with the medical disposable blunt needle to form the tracheal cannula sleeve needle.

In a specific embodiment, the thickness of the medical disposable blunt needle is 18G-30G, preferably 18G-27G; the length of the medical disposable blunt needle is 25-100 mm, preferably 38-70 mm; the length of the outer sleeve of the human radial artery puncture needle is 50-80 percent, preferably 60-70 percent, of the length of the medical disposable blunt needle.

In the present invention, the needle length refers to the length of the metal needle exposed outside the plastic supporting material.

In a specific embodiment, the kit further comprises a plastic box for packaging the tracheal cannula needle, wherein the plastic box is matched with the sizes of a medical disposable blunt needle and a human radial artery puncture needle outer sleeve.

In a specific embodiment, the syringe is 500 μl in size.

In a specific embodiment, the kit further comprises a lung-damaging liquid, an anesthetic, a physiological saline for diluting the anesthetic, an adhesive tape, and a shaver, preferably the lung-damaging liquid is an endotoxin-containing liquid, and preferably the anesthetic is phenobarbital sodium or pentobarbital.

In a specific embodiment, the kit further comprises a disinfectant, preferably a medical iodophor.

In a specific embodiment, the kit further comprises one or more forceps.

An application of the tool kit in preparing an acute lung injury mouse model.

In a specific embodiment, the application process comprises the following steps: anesthesia, neck shaving, mouse fixation, skin incision and tracheal intubation, extraction of a guide core, injection of lung injury liquid, kneading and pressing of mouse chest and neck suturing, and thermal insulation resuscitation.

The tool set can be used for preparing the acute lung injury mouse model efficiently, accurately, safely and cost-effectively, and is considered to have large-scale popularization and application values.

The invention also provides a preparation method of the acute lung injury mouse model, which comprises the following steps: step A, anesthesia: injecting anesthetic into the abdominal cavity of the mouse to anesthetize the mouse; preferably, the anesthetic is phenobarbital sodium or pentobarbital, and is dissolved by normal saline with the concentration of 0.5-2%; step B, neck shaving and mouse fixation: shaving the hair on the neck of the mouse and fixing the limbs of the mouse; preferably, the hair of the neck of the mouse is removed by using a shaving apparatus; step C, skin incision and tracheal intubation: longitudinally incising the neck skin of the mouse, and then pulling the mouse left and right by using a pair of draw hooks to expose the operation field until the trachea is seen, so that the operation is gentle without hurting any neck tissues; then, the tracheal cannula sleeve needle is inserted through the oral cavity of the mouse, and the outer sleeve of the human radial artery puncture needle of the tracheal cannula sleeve needle is clearly observed to enter the trachea at the neck in the inserting process; step D, pulling out the guide core and injecting lung injury liquid: after the intubation is completed, the guide core, namely the medical disposable blunt needle of the tracheal cannula sleeve needle, is pulled out and the drag hook is removed; the mouse is upright and the syringe is used for injecting lung injury liquid into the lung of the mouse through the upper opening of the outer sleeve of the human radial artery puncture needle; preferably, the lung-damaging fluid is an endotoxin-containing liquid; step E, rubbing and pressing the chest of the mouse: after the lung injury liquid injection is finished, the chest of the mouse is rubbed and pressed; step F, neck suture and thermal insulation recovery: the neck skin of the mice was sutured and incubated until the mice wake up.

In the invention, the step E can prevent the injected lung injury liquid from being expectorated out of the airway by the mice and can better diffuse in the lung.

In a specific embodiment, in step B, the mice are placed on the console in a supine position after shaving, and the limbs of the mice are preferably secured by taping.

In a specific embodiment, the step C of skin incision is preceded by a further step of disinfecting the neck skin, preferably using medical iodophors.

In a specific embodiment, the drag hook used in step C is a smooth bipedal drag hook, the two feet of the drag hook are curved and parallel to each other, and the interval between the two feet is 0.5-8 mm, preferably 1-4 mm.

In a specific embodiment, the syringe used in step D is a 500 μl insulin syringe.

In a specific embodiment, in step D, a control group of mice model of acute lung injury is established by injecting PBS buffer to the additional mice in place of lung injury fluid.

In a specific embodiment, the outer cannula of the human radial artery puncture needle is pulled out before or during the step E of kneading the mouse chest cavity.

In a specific embodiment, the total time for rubbing the chest of the mouse in step E is between 10 and 60 seconds, preferably between 20 and 30 seconds.

In a specific embodiment, the neck of each mouse in step F is sutured with two needles.

In a specific embodiment, step F further comprises observing the hair status, behavioral response, respiratory status and body weight of the mouse at a time after cervical suturing.

The tool set and the modeling method have at least the following beneficial effects:

1) The success rate of modeling is high and the efficiency is high. 2) Modeling product mice were more stable in severity of morbidity. 3) The endotoxin consumption in modeling is saved. 4) Is suitable for mice with different body sizes. 5) The operator can get on hand quickly; and the tool set and modeling method can be used for delivering any liquid medicine to the bronchi and lungs of mice, thus having wide application potential. In general, the invention stabilizes the phenotype of the acute lung injury mouse model, improves the model making efficiency and reduces the economic cost.

Drawings

Fig. 1 is a photograph after skin incision in preparing a mouse model of acute lung injury.

Fig. 2 is a photograph showing the exposed trachea after incision of skin using a pair of draw hooks left and right when preparing a mouse model of acute lung injury.

Fig. 3 is a photograph after tracheal intubation in the preparation of a mouse model of acute lung injury.

Fig. 4 is a photograph of the pilot core removed in preparation of a mouse model of acute lung injury.

Fig. 5 is a photograph of endotoxin injection in preparing a mouse model of acute lung injury.

FIG. 6 is a photograph of the chest of a mouse rubbed and pressed in preparation of a mouse model of acute lung injury.

FIG. 7 is another photograph of the chest of a mouse rubbed and pressed in preparation of a mouse model of acute lung injury.

Fig. 8 is a photograph of cervical suturing in preparing a mouse model of acute lung injury.

Fig. 9 is a photograph of a kit for preparing a mouse model of acute lung injury.

Fig. 10 is a photograph of the two-component separation of the tracheal cannula of fig. 9.

Fig. 11 is a schematic view of an endotracheal tube catheter for sale.

FIG. 12 is a graph showing total protein content of mouse alveolar lavage fluid modeled by various modeling methods.

Fig. 13 is a graph showing the wet dry weight ratio of the whole lung of mice modeled by various modeling methods.

FIG. 14 is a graph showing comparison of body weights of mice modeled by various modeling methods.

FIG. 15 shows the concentrations of inflammatory factors in the alveolar lavage fluid of mice modeled by various modeling methods.

FIG. 16 is a comparison of modeling times for modeling by various modeling methods.

FIG. 17 is a graph of lung pathology HE staining of mice modeled by various modeling methods.

Detailed Description

In specific embodiments, the objective is to further establish a stable ARDS/ALI mouse model by improving post-tracheal intubation dosing regimen and optimizing ARDS/ALI model mouse procedure.

The materials used are as follows: endotoxin (LPS), 1% phenobarbital sodium, physiological saline, tracheal cannula (arterial puncture catheters and appropriate cannula guide cores of different specifications) and syringes (specification: 500 ul), retractor, needle, suture, forceps and scissors. In one specific embodiment, the tracheal cannula used in the present invention comprises a medical disposable blunt needle with a needle length of 50mm and a thickness of 18G and a human radial artery puncture needle outer cannula with a needle length of 32mm as the outer cannula of the medical disposable blunt needle. The various types of tracheal cannula needles used in the present invention are commercially available. The tracheal cannula needle was packaged using a plastic box 126mm in length. Specifically, the core of the human radial artery puncture needle is discarded, the outer sleeve thereof is reserved, and a medical disposable blunt needle matched with the outer sleeve of the human radial artery puncture needle is used, and the two are combined to form the tracheal cannula sleeve needle. In addition, the syringe is compatible with a medical disposable blunt needle model.

Animals: C57B wild mice.

Modeling flow:

1. anesthesia: mice were anesthetized with intraperitoneal injection of 1% phenobarbital sodium.

2. Preparation before tracheal intubation: after the mice enter an anesthetic state, the hairs of the necks of the mice are shaved, the mice are placed on an operation table in a supine position, and the mice are fixed by adopting a method of sticking the four limbs of the mice by using adhesive tapes.

3. Tracheal cannula: the neck of the mouse is sterilized by using medical iodophor, the skin of the neck is longitudinally cut, and two draw hooks are used for exposing the operation field until the trachea is hidden, so that the operation is gentle without hurting any neck tissues. Next, the tracheal cannula is inserted through the mouth of the mouse, and the catheter should be clearly observed to enter the trachea at the neck during the insertion process.

4. Endotoxic injection after tracheal intubation: after the intubation was completed, the mice were stood upright and an acute lung injury model was established by injecting LPS (lipopolysaccharide, i.e. endotoxin) or PBS buffer of the control group into the lungs of the mice using a 500 μl insulin syringe. Immediately after the injection, the chest of the mice was rubbed and pressed.

5. Neck suturing and resuscitation: the neck skin of the mouse is sutured, the temperature is kept, and after the mouse wakes up, the hair state, the behavioral response, the respiratory state, the weight and the like of the mouse are observed at any time.

Evaluation of ards/ALI model: respiratory rate, lung pathology index, protein content of alveolar lavage fluid, wet-dry ratio, inflammatory factor, body weight, and mortality of mice.

Respiratory rate: the minute breathing rate 24 hours after the experimental and control mice were modeled was recorded.

Lung pathology index: HE staining observed pathological changes such as inflammation, necrosis, hemorrhage, etc. in lung tissue and alveoli of mice in the experimental group and the control group.

Protein content of alveolar lavage fluid: at 48h of modeling, alveolar lavage fluid from the experimental and control groups was collected, centrifuged, and the supernatant was isolated and assayed for total protein using BCA.

Wet-to-dry ratio: at the time of modeling for 48 hours, lung tissues of the experimental group and the control group are collected, wet weights are weighed and recorded, dry weights are recorded after drying in an oven for 48 hours, and finally wet weight/dry weight is calculated.

Inflammatory factors: ELISA detects alveolar lavage fluid inflammatory factor IL-1 beta.

Weight and mortality: the weight change and mortality were recorded over 7 days in the experimental and control groups.

Fig. 12 to 17 are respectively a total protein content of a mouse alveolar lavage fluid, a wet-dry weight ratio of a whole mouse lung, a comparison graph of a mouse weight, a comparison graph of an inflammatory factor concentration of the mouse alveolar lavage fluid, a comparison of modeling time and a graph of a pathological HE staining of the mouse lung after modeling by various modeling methods. The non-invasive tracheal cannula administration method in fig. 12 to 17 is a tracheal blinding administration method in the background art, and the invasive tracheal cannula administration method is a modeling method proposed in the present invention.

As can be seen from fig. 12 to 17, compared with various ARDS/ALI modeling schemes, the invasive airway administration method provided by the present invention induces a more stable and serious lung injury phenotype under the condition of applying the same dose of lipopolysaccharide (endotoxin), and according to this scheme, the use of part of lipopolysaccharide can be saved, and thus the modeling cost can be saved. In the molding time, the average molding time of the invasive airway administration method is 5 minutes, and compared with the nasal drip method and the noninvasive airway administration method, the molding time is relatively prolonged by 1-2 minutes, but is obviously superior to that of the invasive airway injection method. In terms of model stability, we consider that drug entry into the lungs through the longer respiratory tract is an objective factor for instability of the nasal drop delivery modeling regimen (part of lipopolysaccharide resides or is choked to the outside of the body); the invasive airway internal injection method has long and complex molding time and high molding failure rate, and is a key factor for eliminating the invasive airway internal injection method; in addition, the noninvasive trachea cannula administration method lacks a perfect scheme to identify the problem that the trachea cannula enters the lung and the esophagus, so that the success rate of molding is greatly reduced. The problem of creating a model scheme is that potential incisions possibly induce skin infection and the like exist, however, the modeling method has extremely low infection occurrence rate and cannot influence the whole model through strict procedures of shaving the neck of a mouse, disinfecting and the like.

In summary, the invasive tracheal intubation administration method is considered to be an efficient, accurate, safe and cost-effective molding scheme, and is considered to have large-scale popularization and application values.

In addition, as can be seen from the modeling process and results, the tool set and the modeling method of the invention have at least the following advantages:

1) The success rate of modeling is high and the efficiency is high.

Compared with a surgical trachea cannula method for cutting the trachea of a mouse, the method only needs to cut the skin of the mouse and does not need to cut the trachea; therefore, the invention does not affect the survival rate of mice at all. That is, the invention only opens the skin with a small incision, and the skin cutting has no influence on the survival rate of the mice, so the death rate of the mice is extremely low; the modeling success rate of the invention can almost reach 100 percent.

Compared with a noninvasive trachea cannula administration method, the invention judges whether the cannula is successfully inserted through the oral cavity of the mouse by using the trachea cannula sleeve needle, and the insertion process can clearly observe that the catheter enters the trachea of the mouse after cutting the skin and pulling open the retractor to expose the trachea, namely, the invention can accurately judge whether the cannula is successfully or not only by the operator.

In the prior art, a problem exists in that the success rates of different operators modeling by using the same method are different and even very different. Modeling using the method of the present invention can achieve modeling stability to nearly 100% levels for almost any operator. Second, the modeling methods of the prior art have a success rate that is not comparable to the modeling method of the present invention, even for operators who are very skilled in the operation of a particular method. In addition, the modeling success rate of various modeling methods in the prior art is different for mice with different body types and sizes; even these methods can lead to modeling success or failure due to individual differences in mice. The modeling success rate of the method provided by the invention can be close to 100% by only selecting a tracheal cannula sleeve needle with a proper size no matter what size of the mice, and the modeling method provided by the invention is not influenced by individual differences of the mice.

2) Modeling product mice were more stable in severity of morbidity.

The modeling method provided by the present invention induces a more stable and severe lung injury phenotype using the same dose of lipopolysaccharide (endotoxin).

3) The endotoxin consumption in modeling is saved.

Because the invention induces a more stable and severe lung injury phenotype under the same dosage of lipopolysaccharide, the proposal can save the use of partial lipopolysaccharide and further save the molding cost.

4) Is suitable for mice with different body sizes.

The method is not only suitable for 20-30g mice, but also has no problem in modeling smaller mice. For example, a disposable, blunt needle of 18G thickness and 50mm length can be used to successfully complete tracheal intubation in all mice weighing 14G or more. The tracheal cannula of the mouse with the weight of less than 14g can be realized by the method only by selecting a finer tracheal cannula sleeve needle. Therefore, the method of the present invention has little limitation on the body type of the modeling mouse.

5) The operator can get on hand quickly; and the tool set and modeling method can be used for delivering any liquid medicine to the bronchi and lungs of mice, thus having wide application potential.

The foregoing examples are provided for the purpose of clearly illustrating the technical aspects of the present invention and are not to be construed as limiting the embodiments of the present invention. Any other equivalent technical characteristics may be changed or modified without changing the basic idea and essence of the present invention, and the present invention shall fall within the scope of the claims.

Claims

1. A tool set for preparing a mouse model of acute lung injury, which is characterized by comprising an injector, two draw hooks, a suture needle, a suture, one or more scissors and a tracheal cannula sleeve needle consisting of a medical disposable blunt needle and an outer sleeve of a human radial artery puncture needle matched with the medical disposable blunt needle.

2. The kit of claim 1, wherein the medical disposable blunt needle has a thickness of 18G to 30G, preferably 18G to 27G; the length of the medical disposable blunt needle is 25-100 mm, preferably 38-70 mm; the length of the outer sleeve of the human radial artery puncture needle is 50-80 percent, preferably 60-70 percent, of the length of the medical disposable blunt needle.

3. The kit of claim 1, further comprising a plastic box for packaging the tracheal cannula needle, the plastic box being sized to fit a medical disposable blunt needle and a human radial artery puncture needle outer cannula.

4. The kit of claim 1, wherein the syringe is 500 μl in size.

5. Kit according to any one of claims 1 to 4, further comprising a lung-damaging fluid, an anesthetic, a physiological saline for diluting the anesthetic, a tape, a shaver, preferably the lung-damaging fluid is an endotoxin-containing liquid, preferably the anesthetic is phenobarbital sodium or pentobarbital.

6. Kit according to claim 5, further comprising a sterilizing fluid, preferably said sterilizing fluid is medical iodophor.

7. The kit of claim 5, further comprising one or more forceps.

8. Use of a kit according to any one of claims 1 to 7 in the preparation of a mouse model of acute lung injury.

9. The application according to claim 8, characterized in that the application process comprises the steps of: anesthesia, neck shaving, mouse fixation, skin incision and tracheal intubation, extraction of a guide core, injection of lung injury liquid, kneading and pressing of mouse chest and neck suturing, and thermal insulation resuscitation.