CN116236314A - Improved novel noninvasive mouse trachea instillation molding method - Google Patents
Improved novel noninvasive mouse trachea instillation molding method Download PDFInfo
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Abstract
The invention discloses an improved novel noninvasive mouse trachea instillation molding method, which respectively relates to the processes of manufacturing a mouse suspension fixing plate device, manufacturing a trachea cannula needle, manufacturing a temporary spirometer, performing mouse trachea cannula operation and the like in a bleomycin pulmonary fibrosis model, specifically, suspending and fixing each mouse on a self-made mouse fixing plate after anesthesia, exposing glottis, quantitatively instilling bleomycin solution with the concentration of 5.0mg/mL in the mouse trachea according to the weight of 10 mu L/10G by using a 20G plastic catheter needle, and carrying out SPF-level feeding, wherein the bleomycin-induced pulmonary fibrosis model molding is successful. The method is simple to operate, low in cost, high in success rate of a bleomycin-induced pulmonary fibrosis model and progressive in fibrosis degree. The invention provides a stable and reliable experimental animal model for further researching the pathogenesis of bleomycin-induced pulmonary fibrosis and drug screening.
Description
[ field of technology ]
The invention belongs to the technical field of medicine and experiments, and particularly relates to an improved novel noninvasive mouse trachea instillation molding method.
[ background Art ]
Idiopathic pulmonary fibrosis (Idiopathic Pulmonary Fibrosis, IPF) is a chronic progressive fibrotic interstitial lung disease, typically characterized by excessive production and deposition of extracellular matrix (ECM) and remodeling of abnormal lung tissue structures, and currently there is no cure, with an average age of over 65 years at diagnosis and an average survival time of 2-3 years after diagnosis; it is the most common cause of idiopathic interstitial pneumonia and has previously been considered a rare disease, but the incidence and prevalence is on the rise worldwide. To date, global IPF morbidity is 0.2-93.7/10 ten thousand/year, with higher european and american morbidity and lower asia and south america morbidity. The main clinical manifestations of IPF are progressive exacerbations of dyspnea and reduced lung function. Despite the current increased awareness of IPF and advances in treatment, there is no other method of treating IPF other than lung transplantation.
IPF is an senile disease, and how to prevent and treat idiopathic pulmonary fibrosis has been a worldwide problem. Currently, some drugs such as pirfenidone have been used to treat idiopathic pulmonary fibrosis, but rejection, complications and high treatment costs limit their clinical use. Therefore, there is an urgent need to develop novel high-efficiency anti-idiopathic pulmonary fibrosis drugs, and the development of drugs is now mainly performed on mouse pulmonary fibrosis models.
It is presently believed that IPF is caused by sustained or repeated lung epithelial damage and subsequent activation of fibroblasts and differentiation of myofibroblasts. The sustained myofibroblast phenotype leads to excessive deposition of ECM and abnormal pulmonary repair, leading to formation of tissue scar, distortion of alveolar structure and irreversible loss of lung function. Pulmonary dysfunction caused by pulmonary fibrosis of lung tissue seriously affects and even jeopardizes human life health. Studies have shown that bleomycin-induced pulmonary fibrosis is highly consistent with the progression of idiopathic pulmonary fibrosis, and thus bleomycin-induced murine models of pulmonary fibrosis are generally used.
In the prior art, in order to construct an ideal pulmonary interstitial fibrosis model, the methods for making bleomycin enter the trachea mainly comprise 3 methods:
1. the method comprises the steps of directly injecting an air pipe ring, namely, cutting the neck skin of a mouse by operation, separating muscles and exposing the air pipe, directly injecting bleomycin solution from the air pipe by an injection needle, and suturing a wound to obtain a pulmonary fibrosis model;
2. the atomizer performs atomization inhalation, namely, the atomizer atomizes the bleomycin solution and then automatically inhales the bleomycin solution along with the respiration of the mice;
3. the traditional trachea instillation, namely the trachea cannula is carried out through the mouth and nose of a mouse, a mouse trachea cannula sleeve or a vein retaining needle is generally used as a trachea instillation tool, and the bleomycin solution is instilled into the trachea through the mouth and nose trachea cannula, so that the method is closest to the method of the application.
However, several existing methods still have technical defects:
1. although the airway can be accurately observed by tracheal ring injection, the injury to the mice is larger, the mice are not easy to tolerate, and the mice are easy to be infected and die, so that not only is economic loss caused, but also the follow-up experiment is affected;
2. the effective inhalation of the bleomycin solution is difficult to quantify by an atomizer atomization inhalation method, so that modeling pathological degree is uneven, and the reliability of successful model establishment is greatly reduced; and additional instruments and consumables are required to be used, so that the experimental expenses and time cost are increased.
3. The airway position is difficult to accurately observe in the operation of the traditional tracheal instillation method, and the airway position is easy to be mistakenly inserted into the esophagus to cause failure of molding or damage of the tracheal pharyngeal portion.
[ invention ]
The invention aims to solve the technical problems that: 1. although the airway can be accurately observed by tracheal ring injection, the injury to the mice is larger, the mice are not easy to tolerate, and the mice are easy to be infected and die, so that not only is economic loss caused, but also the follow-up experiment is affected; 2. the effective inhalation of the bleomycin solution is difficult to quantify by an atomizer atomization inhalation method, so that modeling pathological degree is uneven, and the reliability of successful model establishment is greatly reduced; in addition, extra instruments and consumables are needed, so that the experiment expense and the time cost are increased; 3. the airway position is difficult to accurately observe in the operation of the traditional tracheal instillation method, and the airway position is easy to be mistakenly inserted into the esophagus to cause failure of molding or damage of the tracheal pharyngeal portion. Aiming at the technical problems, the invention provides an improved novel noninvasive mouse trachea instillation molding method, which is an improved noninvasive mouse trachea instillation molding method, and aims to enable a mouse trachea instillation experiment to be simpler and more convenient, greatly improve success rate of mouse pulmonary fibrosis molding and survival rate of mice, and provide stable and reliable technical support for further researching pathogenesis of pulmonary fibrosis and screening medicines for treating pulmonary fibrosis.
The aim of the invention is achieved by the following technical scheme:
an improved novel noninvasive mouse trachea instillation molding method comprises the following steps:
s1, preparation before operation:
s1-1: sterilizing the 100 mu L gun head, the plastic flat-headed pliers, the tongue depressor and the 20G plastic catheter needle by an autoclave;
s1-2: preparing bleomycin solution by using a biosafety cabinet, and ensuring uniform mixing;
s1-3: preparing a clean workspace comprising 1m 2 Is a working space of (2);
s1-4: a self-made mouse hanging fixing plate is opened and erected on a workbench in front of an operator;
s1-5: tying a single-length 10cm No. 4.0 suture on a top screw of the operation plate;
s1-6: loosely fixing the temporary pneumotometer of the self-made mouse on a 20G plastic catheter needle, and placing the temporary pneumotometer on one side of a hanging fixing plate of the self-made mouse for later use;
s1-7: extracting 300 mu L of air into a 1mL syringe, and placing the syringe on one side of a self-made mouse hanging fixing plate for later use;
s1-8: cutting a piece of medical adhesive tape with the length of 14cm, and placing the medical adhesive tape on one side of the plate for standby, wherein the medical adhesive tape is used for fixing mice on the plate;
s1-9: 20% (w/v) uratam was prepared, and the anesthetic was taken from the mice by intraperitoneal injection according to a weight of 0.8mL/100g, and the depth of anesthesia was observed;
s2, trachea cannula:
s2-1: when waiting for the anesthesia effect of the mice, a 100 mu L gun head is arranged by a liquid-transferring gun, and the bleomycin solution with the concentration of 5.0mg/mL is sucked according to the weight of 10 mu L/10g and is placed on one side of the plate for standby;
s2-2: after the preparation, hanging the upper incisors of the mice on a wire for fixing the screws of the operation platform, so as to ensure that the mice are in a supine position and the backs of the mice lie on the surface of the platform;
s2-3: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse and cannot be fixed too tightly so as not to limit respiration;
s2-4: the flashlight is turned on to be fixed at a distance of 1 cm to 2cm from the surface of the skin at the position of the trachea;
s2-5: fixing the tongue by using sterile flat-headed forceps; carefully avoid the lower incisors, gently grasp the tongue, withdraw the tongue from the mouth;
s2-6: inserting the tongue depressor with the remaining hand, flattening the tongue on the bottom of the mouth, releasing the forceps, and holding the tongue depressor stationary in the next two steps;
s2-7: guiding the flashlight to the near position until reaching the level of the main bronchus, and determining the direction of lamplight so as to be capable of seeing the condition of the bronchus;
s2-8: tilting the syringe to follow the natural path of the trachea, and placing the tip of the 20G plastic catheter directly into the lumen with a temporary spirometer containing PBS droplets which will rise and fall with each breath after placement is successful;
s2-9: feeding the 20G plastic catheter into the tongue depressor for 5mm, and taking out the tongue depressor;
s2-10: the syringe is moved to the other hand, the pintle is grasped, and the syringe is gently taken out;
s2-11: injecting bleomycin solution into the inner center of the plastic catheter pintle, connecting a second injector after the solution flows in, and rapidly injecting 300ul of air into the plastic catheter;
s2-12: replacing the second syringe with the first syringe containing the PBS droplet, wherein if the tracheal intubation is successfully performed, the droplet can continuously rise and fall;
s3, postoperative care:
placing the mice on an operating table, allowing the bleomycin solution to enter the lung, taking out the plastic catheter and the adhesive tape, placing the mice in a dry and warm place, closely observing the respiration state of the mice and monitoring the heart rate by a stethoscope until the mice are recovered to be normally fed in a cage after waking up, observing the state of the mice every day, and weighing the weight;
s4, taking lung tissues of the mice to prepare pathological sections, and observing after staining:
after 28 days of raising, 20% (w/v) uratam solution is injected into the abdominal cavity, after anesthesia, the mice are directly sacrificed by cervical dislocation, lung tissues are taken out after thoracotomy and photographed, the left lung of the mice is fixed by 4% (v/v) paraformaldehyde, then the mice are dehydrated by ethanol, waxed, sliced by a slicer of 3 mu m, dewaxed by dimethylbenzene, H & E staining process, the structural change of the lung tissues is observed under a microscope, and finally pathological result analysis is carried out.
In the invention, the following components are added:
the self-made temporary spirometer for mice described in step S1-6 is characterized in that a plunger in a 1mL syringe is taken out, the plunger is discarded, and 60 mu L PBS is stored at the top of the syringe to form a closed space, so that the temporary spirometer is produced, and the purpose of the temporary spirometer is to ensure that a cannula is inserted into a trachea rather than an esophagus, and the success of operation can be judged after the bleomycin solution is instilled.
The self-made mouse hanging fixing plate in the step S1 is made of wood, the fixing plate and the workbench generate isosceles triangles with base angles of 60-80 degrees, an inclined operation interface is arranged, a bottom supporting surface and a side cross section are arranged, and a number 4.0 line is horizontally arranged above the bottom supporting surface and the side cross section.
The prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse in the step S2-3, and the fixed position and the tightness are noted, so that a certain chest movement range of the mouse is ensured.
The condition of seeing the trachea in the step S2-7 is that the flashlight irradiates the neck of the mouse during the trachea instillation, the trachea can be easily distinguished through the action of breathing, because the breathing can cause the intensity of emitted light to fluctuate, when the position is correct, the condition is distinguished as a centrally-located optical needle on the axial plane, and the ambient light of the oral cavity is minimum.
The rapid injection of 300 μl of air into the plastic catheter described in step S2-11 is convenient for supplementing oxygen to mice and rapidly dispersing bleomycin solution into each bronchiole, so as to prevent choking death in the main bronchi and reduce operation choking death rate.
In the steps S2 and S3, the inner diameter of the 20G plastic catheter needle is about 0.58mm; the 20G plastic catheter needle may also be replaced entirely with a 16G plastic catheter needle or a 23G plastic catheter needle.
And (3) standing the mice on an operating table for 10min in the step (S3).
The bleomycin solution is introduced into the lung in step S3, wherein the total volume of the bleomycin solution injected into the trachea of the mouse is between 20 and 30 mu L, so as to prevent excessive pipetting deviation and asphyxia caused by larger volume.
Compared with the prior art, the invention has the following advantages:
1. the improved novel noninvasive mouse trachea instillation molding method is exquisite in design, firstly, the self-made mouse hanging fixing plate enables the mouse to be placed in a supine hanging mode, the neck and the trunk of the mouse are in a straight line, glottic cracks of the mouse can be conveniently and clearly observed, and guarantee is provided for the accuracy of trachea cannula; secondly, the self-made plastic trachea cannula needle can effectively ensure that the trachea of the mouse is not damaged, is convenient to operate, greatly reduces death of the mouse caused by traumatic infection, and reduces experimental consumables; thirdly, the self-made temporary vital capacity meter of the mice can ensure the success of the tracheal intubation before instillation through the fluctuation of the liquid level of the temporary vital capacity meter of the self-made mice, thereby greatly reducing the probability of misplugging esophagus; on the other hand, after the bleomycin solution is instilled, the operation process is further verified that the cannula needle is not separated from the trachea and the medicine is successfully injected into the trachea, so that the success rate of molding is greatly improved.
2. The improved novel noninvasive mouse tracheal instillation molding method successfully establishes a bleomycin pulmonary fibrosis mouse model by quantitatively instilling bleomycin solution in the trachea, and the pulmonary fibrosis degree of the bleomycin pulmonary fibrosis mouse model is gradually aggravated along with the prolongation of molding time within one month and is expressed as progressive pulmonary fibrosis; the lung fibrosis model obtained by the method has the advantages of high success rate, uniform pathological degree, short experimental period, low cost, simple and convenient operation, high success rate of the mouse lung fibrosis model constructed by the method and low death rate of the mouse; provides stable and reliable technical conditions for further experimental study, and is helpful for study of the pathogenesis of bleomycin-induced pulmonary fibrosis and drug screening.
[ description of the drawings ]
FIG. 1 is a flow chart of an experimental procedure of a modified novel noninvasive mouse tracheal instillation molding method and preparation device according to the present invention;
FIG. 2 is a chart of the trachea of a bleomycin-induced pulmonary fibrosis mouse model in trachea instillation constructed in the invention;
FIG. 3 is a graph of the liquid surface fluctuation in the instillation of a homemade temporary spirometer and trachea for a bleomycin-induced pulmonary fibrosis mouse model constructed in accordance with the present invention;
FIG. 4 is a diagram of a self-made mouse suspension fixation plate device for a bleomycin-induced pulmonary fibrosis mouse model constructed in accordance with the present invention;
FIG. 5 is a graph showing the appearance of lung tissue of a bleomycin-induced pulmonary fibrosis mouse model group and a normal group mouse constructed in example 1 of the present invention;
FIG. 6 is a chart showing the pathology of H & E staining of lung tissue sections of bleomycin-induced pulmonary fibrosis mice model group and normal group mice constructed in example 1 of the present invention.
[ detailed description ] of the invention
The following describes the invention in more detail with reference to examples. The following examples are intended to illustrate the invention and are not intended to be limiting. The experimental examples were conducted in accordance with the conventional methods without specifying the specific conditions.
Example 1:
30 SPF-class male mice, the weight of which is 23+/-2 g, are purchased from animal experiment centers of university of Anhui medical science, and then are directly fed to SPF-class laboratory centers of animal centers of medical college of Right river, all the experiment mice are living in a specific sterile environment, the temperature is controlled to be 25+/-2 ℃, the humidity is controlled to be 60+/-10%, the bright-dark period is 12h/12h, and the conditions allow the mice to eat and drink water freely. After one week of feeding, the animals were randomly divided into model and normal groups. In addition, all procedures of this experiment were agreed by the biological research ethics committee of the national medical college of right river.
An improved novel noninvasive mouse trachea instillation molding method comprises the following steps:
s1, preoperative preparation
Step 1-1: sterilizing the 100 mu L gun head, the plastic flat-headed pliers, the tongue depressor and the 20G plastic catheter needle by an autoclave;
step 1-2: preparing bleomycin solution by using a biosafety cabinet, and ensuring uniform mixing;
step 1-3: preparing a clean workspace comprising 1m 2 Is a working space of (2);
step 1-4: the self-made mouse hanging fixing plate is opened and erected on a workbench in front of an operator;
step 1-5: a suture No. 4.0 with a single length of 10cm is tied on a screw at the top end of the operation plate;
step 1-6: loosely fixing a self-made mouse temporary spirometer on a 20G plastic catheter needle, and placing the self-made mouse temporary spirometer on one side of a fixing plate for later use;
step 1-7: draw 300 μl of air into a 1mL syringe, place on one side of the plate for use;
step 1-8: cutting a piece of medical adhesive tape with the length of 14cm, placing the medical adhesive tape on one side of the plate for standby, and fixing the mice on the plate;
step 1-9: 20% (w/v) uratam was prepared, and a mouse weighing 25g was intraperitoneally injected with a solution of 0.20mL uratam according to a weight of 0.8mL/100g, and the depth of anesthesia was observed;
s2, trachea cannula
Step 2-1: when waiting for the anesthesia effect of the mice, a 100 mu L gun head is arranged on a liquid-transfering gun to suck 25 mu L of bleomycin solution with the concentration of 5.0mg/mL, and the bleomycin solution is placed on one side of a plate for standby;
step 2-2: after the preparation, hanging the upper incisors of the mice on a wire for fixing the screws of the operation platform, so as to ensure that the mice are in a supine position and the backs of the mice lie on the surface of the platform;
step 2-3: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse and cannot be fixed too tightly so as not to limit respiration;
step 2-4: the flashlight is turned on to be fixed at a distance of 1 cm to 2cm from the skin surface at the tracheal position;
step 2-5: fixing the tongue with sterile flat-headed forceps, carefully avoiding the lower incisors, gently grasping the tongue, and withdrawing the tongue from the oral cavity;
step 2-6: the remaining hand is used to insert the self-making tongue depressor, which is used to flatten the tongue against the bottom of the mouth, releasing the forceps, but holding the tongue depressor stationary in the next two steps;
step 2-7: by directing the flashlight proximally until it reaches the level of the main bronchi, the direction of the light is determined so that the condition of the bronchi can be seen;
step 2-8: tilting the syringe to follow the natural path of the trachea and placing the 20G plastic catheter tip directly into the lumen with the temporary spirometer containing droplets, after placement is successful, the PBS droplets will begin to rise and fall with each breath;
step 2-9: feeding the 20G plastic catheter into the tongue depressor for 5mm, and taking out the tongue depressor;
step 2-10: the syringe is moved to the other hand, the pintle is grasped, and the syringe is gently taken out;
step 2-11: injecting bleomycin solution into the inner center of the plastic catheter pintle, connecting a second injector after the solution flows in, and rapidly injecting 300 mu L of air into the plastic catheter;
step 2-12: replacing the second syringe with the first syringe containing the PBS droplet, wherein if the tracheal intubation is successfully performed, the droplet can continuously rise and fall;
s3, postoperative care
Placing the mice on an operating table for 10min to facilitate the bleomycin solution to enter the lung, taking out the plastic catheter and the adhesive tape, placing the mice in a dry and warm place, closely observing the respiration state of the mice and monitoring the heart rate by a stethoscope until the mice are recovered to be normally fed in a cage after waking up, observing the state of the mice every day, and weighing the weight;
s4, taking lung tissues of the mice to manufacture pathological sections, and observing after staining:
after 28 days of raising, 20% (w/v) uratam solution is injected into the abdominal cavity, after anesthesia, the mice are directly sacrificed by cervical dislocation, lung tissues are taken out after thoracotomy and photographed, the left lung of the mice is fixed by 4% (v/v) paraformaldehyde, then the mice are dehydrated by ethanol, waxed, sliced by a slicer of 3 mu m, dewaxed by dimethylbenzene, H & E staining process, the structural change of the lung tissues is observed under a microscope, and finally pathological result analysis is carried out.
FIG. 2 is a chart of the trachea in a constructed bleomycin-induced pulmonary fibrosis mouse model trachea instillation;
FIG. 3 is a graph of the liquid surface fluctuation in a self-made temporary spirometer and tracheal instillation used in constructing a bleomycin-induced pulmonary fibrosis mouse model;
FIG. 4 is a diagram of a self-made mouse suspension fixation plate device for use in constructing a bleomycin-induced pulmonary fibrosis mouse model;
FIG. 5 is a graph showing the appearance of lung tissues of a bleomycin-induced pulmonary fibrosis mouse model group and a normal group mouse constructed in example 1, wherein the lung tissues of the mice in the model group are white in color, hard in texture, with white nodules protruding out of the lung surface, red in the lung tissues of the normal group and soft in texture after being fed for 28 days through the air tube instillation of bleomycin as shown in FIG. 5;
FIG. 6 is a graph showing the pathology of H & E staining of lung tissue sections of bleomycin-induced lung fibrosis mice model group and normal group mice constructed in example 1. As shown in FIG. 6, H & E staining shows that mice in model group have a large number of disordered alveolar structures, thickened alveolar spaces, collapsed alveolar cavities, inflammatory cell infiltration and severe lung fibrosis, severe, almost complete disappearance of lung tissue structure destruction and severe lesions compared with normal group. The alveoli of the normal group of mice are composed of single-layer cells, and the whole lung tissue structure is clear, has no inflammatory cell infiltration and no fibrosis nodule formation.
Example 2:
30 SPF-class male mice, the weight of which is 23+/-2 g, are purchased from animal experiment centers of university of Anhui medical science, and then are directly fed to SPF-class laboratory centers of animal centers of medical college of Right river, all the experiment mice are living in a specific sterile environment, the temperature is controlled to be 25+/-2 ℃, the humidity is controlled to be 60+/-10%, the bright-dark period is 12h/12h, and the conditions allow the mice to eat and drink water freely. After one week of feeding, the animals were randomly divided into model and normal groups. In addition, all procedures of this experiment were agreed by the biological research ethics committee of the national medical college of right river.
An improved novel noninvasive mouse trachea instillation molding method comprises the following steps:
s1, preoperative preparation
Step 1-1: sterilizing the 100 mu L gun head, the plastic flat-headed pliers, the tongue depressor and the 23G plastic catheter needle by an autoclave;
step 1-2: preparing bleomycin solution by using a biosafety cabinet, and ensuring uniform mixing;
step 1-3: preparing a clean workspace comprising 1m 2 Is a working space of (2);
step 1-4: the self-made mouse hanging fixing plate is opened and erected on a workbench in front of an operator;
step 1-5: a suture No. 4.0 with a single length of 10cm is tied on a screw at the top end of the operation plate;
step 1-6: loosely fixing a self-made mouse temporary spirometer on a 23G plastic catheter needle, and placing the self-made mouse temporary spirometer on one side of a fixing plate for later use;
step 1-7: draw 300 μl of air into a 1mL syringe, place on one side of the plate for use;
step 1-8: cutting a piece of medical adhesive tape with the length of 14cm, placing the medical adhesive tape on one side of the plate for standby, and fixing the mice on the plate;
step 1-9: 20% (w/v) uratam was prepared, and a mouse weighing 25g was intraperitoneally injected with a solution of 0.20mL uratam according to a weight of 0.8mL/100g, and the depth of anesthesia was observed;
s2, trachea cannula
Step 2-1: when waiting for the anesthesia effect of the mice, a 100 mu L gun head is arranged on a liquid-transfering gun to suck 25 mu L of bleomycin solution with the concentration of 5.0mg/mL, and the bleomycin solution is placed on one side of a plate for standby;
step 2-2: after the preparation, hanging the upper incisors of the mice on a wire for fixing the screws of the operation platform, so as to ensure that the mice are in a supine position and the backs of the mice lie on the surface of the platform;
step 2-3: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse and cannot be fixed too tightly so as not to limit respiration;
step 2-4: the flashlight is turned on to be fixed at a distance of 1 cm to 2cm from the skin surface at the tracheal position;
step 2-5: fixing the tongue with sterile flat-headed forceps, carefully avoiding the lower incisors, gently grasping the tongue, and withdrawing the tongue from the oral cavity;
step 2-6: the remaining hand is used to insert the self-making tongue depressor, which is used to flatten the tongue against the bottom of the mouth, releasing the forceps, but holding the tongue depressor stationary in the next two steps;
step 2-7: by directing the flashlight proximally until it reaches the level of the main bronchi, the direction of the light is determined so that the condition of the bronchi can be seen;
step 2-8: tilting the syringe to follow the natural path of the trachea and placing the 23G plastic catheter tip directly into the lumen with the temporary spirometer containing droplets, after placement is successful, the PBS droplets will begin to rise and fall with each breath;
step 2-9: feeding the 23G plastic catheter into the tongue depressor for 5mm, and taking out the tongue depressor;
step 2-10: the syringe is moved to the other hand, the pintle is grasped, and the syringe is gently taken out;
step 2-11: injecting bleomycin solution into the inner center of the plastic catheter pintle, connecting a second injector after the solution flows in, and rapidly injecting 300ul of air into the plastic catheter;
step 2-12: replacing the second syringe with the first syringe containing the PBS droplet, wherein if the tracheal intubation is successfully performed, the droplet can continuously rise and fall;
s3, postoperative care
Placing the mice on an operating table for 10min to facilitate the bleomycin solution to enter the lung, taking out the plastic catheter and the adhesive tape, placing the mice in a dry and warm place, closely observing the respiration state of the mice and monitoring the heart rate by a stethoscope until the mice are recovered to be normally fed in a cage after waking up, observing the state of the mice every day, and weighing the weight;
s4, taking lung tissues of the mice to manufacture pathological sections, and observing after staining:
after 28 days of raising, 20% (w/v) uratam solution is injected into the abdominal cavity, after anesthesia, the mice are directly sacrificed by cervical dislocation, lung tissues are taken out after thoracotomy and photographed, the left lung of the mice is fixed by 4% (v/v) paraformaldehyde, then the mice are dehydrated by ethanol, waxed, sliced by a slicer of 3 mu m, dewaxed by dimethylbenzene, H & E staining process, the structural change of the lung tissues is observed under a microscope, and finally pathological result analysis is carried out.
The modeling of example 2 was performed as provided in example 1, and the resulting model and normal mice had lung tissue morphology and H & E staining pathology patterns of lung tissue sections similar to example 1.
Example 3:
30 SPF-class male mice, the weight of which is 23+/-2 g, are purchased from animal experiment centers of university of Anhui medical science, and then are directly fed to SPF-class laboratory centers of animal centers of medical college of Right river, all the experiment mice are living in a specific sterile environment, the temperature is controlled to be 25+/-2 ℃, the humidity is controlled to be 60+/-10%, the bright-dark period is 12h/12h, and the conditions allow the mice to eat and drink water freely. After one week of feeding, the animals were randomly divided into model and normal groups. In addition, all procedures of this experiment were agreed by the biological research ethics committee of the national medical college of right river.
An improved novel noninvasive mouse trachea instillation molding method comprises the following steps:
s1, preoperative preparation
Step 1-1: sterilizing the 100 mu L gun head, the plastic flat-headed pliers, the tongue depressor and the 16G plastic catheter needle by an autoclave;
step 1-2: preparing bleomycin solution by using a biosafety cabinet, and ensuring uniform mixing;
step 1-3: preparing a clean workspace comprising 1m 2 Is a working space of (2);
step 1-4: the self-made mouse hanging fixing plate is opened and erected on a workbench in front of an operator;
step 1-5: a suture No. 4.0 with a single length of 10cm is tied on a screw at the top end of the operation plate;
step 1-6: loosely fixing a self-made mouse temporary spirometer on a 16G plastic catheter needle, and placing the self-made mouse temporary spirometer on one side of a fixing plate for later use;
step 1-7: draw 300 μl of air into a 1mL syringe, place on one side of the plate for use;
step 1-8: cutting a piece of medical adhesive tape with the length of 14cm, placing the medical adhesive tape on one side of the plate for standby, and fixing the mice on the plate;
step 1-9: 20% (w/v) uratam was prepared, and a mouse weighing 25g was intraperitoneally injected with a solution of 0.20mL uratam according to a weight of 0.8mL/100g, and the depth of anesthesia was observed;
s2, trachea cannula
Step 2-1: when waiting for the anesthesia effect of the mice, a 100 mu L gun head is arranged on a liquid-transfering gun to suck 25 mu L of bleomycin solution with the concentration of 5.0mg/mL, and the bleomycin solution is placed on one side of a plate for standby;
step 2-2: after the preparation, hanging the upper incisors of the mice on a wire for fixing the screws of the operation platform, so as to ensure that the mice are in a supine position and the backs of the mice lie on the surface of the platform;
step 2-3: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse and cannot be fixed too tightly so as not to limit respiration;
step 2-4: the flashlight is turned on to be fixed at a distance of 1 cm to 2cm from the skin surface at the tracheal position;
step 2-5: fixing the tongue with sterile flat-headed forceps, carefully avoiding the lower incisors, gently grasping the tongue, and withdrawing the tongue from the oral cavity;
step 2-6: the remaining hand is used to insert the self-making tongue depressor, which is used to flatten the tongue against the bottom of the mouth, releasing the forceps, but holding the tongue depressor stationary in the next two steps;
step 2-7: by directing the flashlight proximally until it reaches the level of the main bronchi, the direction of the light is determined so that the condition of the bronchi can be seen;
step 2-8: tilting the syringe to follow the natural path of the trachea and placing the 16G plastic catheter tip directly into the lumen with the temporary spirometer containing droplets, after placement is successful, the PBS droplets will begin to rise and fall with each breath;
step 2-9: feeding the 16G plastic catheter into the tongue depressor for 5mm, and taking out the tongue depressor;
step 2-10: the syringe is moved to the other hand, the pintle is grasped, and the syringe is gently taken out;
step 2-11: injecting bleomycin solution into the inner center of the plastic catheter pintle, connecting a second injector after the solution flows in, and rapidly injecting 300ul of air into the plastic catheter;
step 2-12: replacing the second syringe with the first syringe containing the PBS droplet, wherein if the tracheal intubation is successfully performed, the droplet can continuously rise and fall;
s3, postoperative care
The mice are placed on an operating table for 10min, bleomycin solution is facilitated to enter the lung, a plastic catheter and an adhesive tape are taken out, the mice are placed in a dry and warm place, the respiration state of the mice is closely observed, the stethoscope monitors the heart rate until the mice are recovered to be normally fed in a cage after waking up, the state of the mice is observed every day, and the weight is weighed.
S4, taking lung tissues of the mice to manufacture pathological sections, and observing after staining:
after 28 days of raising, 20% (w/v) uratam solution is injected into the abdominal cavity, after anesthesia, the mice are directly sacrificed by cervical dislocation, lung tissues are taken out after thoracotomy and photographed, the left lung of the mice is fixed by 4% (v/v) paraformaldehyde, then the mice are dehydrated by ethanol, waxed, sliced by a slicer of 3 mu m, dewaxed by dimethylbenzene, H & E staining process, the structural change of the lung tissues is observed under a microscope, and finally pathological result analysis is carried out.
The modeling of example 3 was performed as provided in example 1, and the resulting model and normal mice had lung tissue morphology and H & E staining pathology patterns of lung tissue sections similar to example 1.
Comparative example:
30 SPF-class male mice, the weight of which is 23+/-2 g, are purchased from animal experiment centers of university of Anhui medical science, and then are directly fed to SPF-class laboratory centers of animal centers of medical college of Right river, all the experiment mice are living in a specific sterile environment, the temperature is controlled to be 25+/-2 ℃, the humidity is controlled to be 60+/-10%, the bright-dark period is 12h/12h, and the conditions allow the mice to eat and drink water freely. After one week of feeding, the animals were randomly divided into model and normal groups. In addition, all procedures of this experiment were agreed by the biological research ethics committee of the national medical college of right river.
S1, preoperative preparation
Step 1-1: sterilizing the plastic flat-headed pliers, the tongue depressor and the No. 8 straight-headed mouse stomach-lavage needle by an autoclave;
step 1-2: preparing bleomycin solution by using a biosafety cabinet, and ensuring uniform mixing;
step 1-3: preparing a clean workspace comprising 1m 2 Is a working space of (2);
step 1-4: the self-made mouse hanging fixing plate is opened and erected on a workbench in front of an operator;
step 1-5: a suture No. 4.0 with a single length of 10cm is tied on a screw at the top end of the operation plate;
step 1-6: the 1mL syringe needle cylinder is fixed on a No. 8 straight mouse stomach-lavage needle and is placed on one side of a fixing plate for standby;
step 1-7: cutting a piece of medical adhesive tape with the length of 14cm, placing the medical adhesive tape on one side of the plate for standby, and fixing the mice on the plate;
s2, trachea cannula
Step 2-1: a1 mL syringe draws a bleomycin solution at a concentration of 5.0mg/mL at a weight of 10. Mu.L/10 g and places it on one side of the plate for use;
step 2-2: after the preparation, hanging the upper incisors of the mice on a wire for fixing the screws of the operation platform, so as to ensure that the mice are in a supine position and the backs of the mice lie on the surface of the platform;
step 2-3: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse and cannot be fixed too tightly so as not to limit respiration;
step 2-4: the flashlight is turned on to be fixed at a distance of 1 cm to 2cm from the skin surface at the tracheal position;
step 2-5: fixing the tongue with sterile flat-headed forceps, carefully avoiding the lower incisors, gently grasping the tongue, and withdrawing the tongue from the oral cavity;
step 2-6: the remaining hand is used to insert the self-making tongue depressor, which is used to flatten the tongue against the bottom of the mouth, releasing the forceps, but holding the tongue depressor stationary in the next two steps;
step 2-7: by directing the flashlight proximally until it reaches the level of the main bronchi, the direction of the light is determined so that the condition of the bronchi can be seen;
step 2-8: tilting the injector to enable the injector to follow the natural path of the trachea, and directly placing the gastric lavage needle of the No. 8 straight-head mouse into the lumen;
s3, postoperative care
The mice are placed on an operating table for 10min, bleomycin solution is facilitated to enter the lung, a plastic catheter and an adhesive tape are taken out, the mice are placed in a dry and warm place, the respiration state of the mice is closely observed, the stethoscope monitors the heart rate until the mice are recovered to be normally fed in a cage after waking up, the state of the mice is observed every day, and the weight is weighed.
S4, taking lung tissues of the mice to manufacture pathological sections, and observing after staining:
after 28 days of raising, 20% (w/v) uratam solution is injected into the abdominal cavity, after anesthesia, the mice are directly sacrificed by cervical dislocation, lung tissues are taken out after thoracotomy and photographed, the left lung of the mice is fixed by 4% (v/v) paraformaldehyde, then the mice are dehydrated by ethanol, waxed, sliced by a slicer of 3 mu m, dewaxed by dimethylbenzene, H & E staining process, the structural change of the lung tissues is observed under a microscope, and finally pathological result analysis is carried out.
Results:
the success rate of the comparative modeling method is extremely low, and the H & E staining pathological patterns of the lung tissue appearance morphology of the model group and the normal group mice obtained by the mice with successful modeling are similar to those of the example 1; the model group obtained from the mice with the model failure and the lung tissue appearance form of the normal group mice have no difference in H & E staining pathology of the lung tissue sections.
Results and summary:
1. the size of the inner diameter and the outer diameter of the 20G plastic catheter needle in the embodiment 1 is compared with that in the embodiment 2 and the embodiment 3, which are more in line with the model of the mouse trachea, and the operation is simple and the success rate is high;
2. the size of the inner diameter and the outer diameter of the 23G plastic catheter needle of the example 2 is smaller than that of the example 1, and the fluctuation of the liquid level of the temporary spirometer during operation is not obvious so that an operator cannot easily judge whether to insert a trachea or not, and finally the operation time of trachea cannula is prolonged, and the breathing state of a mouse is influenced to a certain extent;
3. the size of the inner diameter and the outer diameter of the 16G plastic catheter needle of the example 3 is larger than that of the example 1, so that the air tube orifice of a mouse is almost blocked, the injection is inconvenient to be deeply inserted into a lumen, and if the hand of an operator is jogged to cause that the air tube orifice of the mouse cannot be attached, part of the finally injected bleomycin solution flows into the esophagus;
4. the comparative examples have a greater disadvantage than examples 1-3: (1) the mice are not subjected to intraperitoneal injection anesthesia, and the operation is carried out in a waking state, so that the influence of anesthesia on the respiratory rate can be eliminated, but the greater difficulty is that the mice are extremely not matched, so that whether the mice are mistakenly inserted into the esophagus or not cannot be distinguished in the operation process; (2) the gastric lavage needle of the No. 8 straight-headed mouse is easy to damage the trachea of the mouse; (3) the syringe absorbs the bleomycin solution and cannot accurately quantify.
Therefore, the lung fibrosis model obtained by the method has the advantages of high success rate, uniform pathological degree, short experimental period, low cost, simple and convenient operation, and the mouse lung fibrosis model constructed by the method has the advantages of high success rate and low death rate.
The invention has been described in further detail in connection with the specific embodiments, but the invention is not limited thereto. Variations or substitutions of technical features of the present technical solution will be considered to be within the scope of the present invention by those of ordinary skill in the art to which the present invention pertains without departing from the inventive concept.
Claims (7)
1. An improved novel noninvasive mouse trachea instillation molding method is characterized in that: the method comprises the following steps:
s1, preparation before operation:
s1-1: sterilizing the 100 mu L gun head, the plastic flat-headed pliers, the tongue depressor and the 20G plastic catheter needle by an autoclave;
s1-2: preparing bleomycin solution by using a biosafety cabinet, and ensuring uniform mixing;
s1-3: preparing a clean workspace comprising 1m 2 Is a working space of (2);
s1-4: a self-made mouse hanging fixing plate is opened and erected on a workbench in front of an operator;
s1-5: tying a single-length 10cm No. 4.0 suture on a top screw of the operation plate;
s1-6: loosely fixing the temporary pneumotometer of the self-made mouse on a 20G plastic catheter needle, and placing the temporary pneumotometer on one side of a hanging fixing plate of the self-made mouse for later use;
s1-7: extracting 300 mu L of air into a 1mL syringe, and placing the syringe on one side of a self-made mouse hanging fixing plate for later use;
s1-8: cutting a piece of medical adhesive tape with the length of 14cm, and placing the medical adhesive tape on one side of the plate for standby, wherein the medical adhesive tape is used for fixing mice on the plate;
s1-9: 20% (w/v) uratam was prepared, and the anesthetic was taken from the mice by intraperitoneal injection according to a weight of 0.8mL/100g, and the depth of anesthesia was observed;
s2, trachea cannula:
s2-1: when waiting for the anesthesia effect of the mice, a 100 mu L gun head is arranged by a liquid-transferring gun, and the bleomycin solution with the concentration of 5.0mg/mL is sucked according to the weight of 10 mu L/10g and is placed on one side of the plate for standby;
s2-2: after the preparation, the upper incisors of the anesthetized mice are hung on the wires for fixing the screws of the operation platform, so that the mice are ensured to be in a supine position, and the backs of the mice lie on the surface of the platform;
s2-3: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse and cannot be fixed too tightly so as not to limit respiration;
s2-4: the flashlight is turned on to be fixed at a distance of 1 cm to 2cm from the surface of the skin at the position of the trachea;
s2-5: fixing the tongue by using sterile flat-headed forceps; carefully avoid the lower incisors, gently grasp the tongue, withdraw the tongue from the mouth;
s2-6: inserting the tongue depressor with the remaining hand, flattening the tongue on the bottom of the mouth, releasing the forceps, and holding the tongue depressor stationary in the next two steps;
s2-7: guiding the flashlight to the near position until reaching the level of the main bronchus, and determining the direction of lamplight so as to be capable of seeing the condition of the bronchus;
s2-8: tilting the syringe to follow the natural path of the trachea, and placing the tip of the 20G plastic catheter directly into the lumen of the trachea with a temporary spirometer containing PBS droplets which will rise and fall with each breath after placement is successful;
s2-9: delivering the 20G catheter into the tongue depressor for 5mm, and taking out the tongue depressor;
s2-10: the syringe is moved to the other hand, the pintle is grasped, and the syringe is gently taken out;
s2-11: injecting bleomycin solution into the inner center of the plastic catheter pintle, connecting a second injector after the solution flows in, and rapidly injecting 300 mu L of air into the plastic catheter;
s2-12: replacing the second syringe with the first syringe containing the PBS droplet, wherein if the tracheal intubation is successfully performed, the droplet can continuously rise and fall;
s3, postoperative care:
placing the mice on an operating table, allowing the bleomycin solution to enter the lung, taking out the plastic catheter and the adhesive tape, placing the mice in a dry and warm place, closely observing the respiration state of the mice and monitoring the heart rate by a stethoscope until the mice are recovered to be normally fed in a cage after waking up, observing the state of the mice every day, and weighing the weight;
s4, taking lung tissues of the mice to manufacture pathological sections, and observing after staining:
after 28 days of raising, 20% w/v uratam solution is injected into the abdominal cavity, the mice are killed directly by cervical dislocation after anesthesia, lung tissues are taken out after thoracotomy and photographed, the left lung of the mice is fixed by 4% v/v paraformaldehyde, then the mice are dehydrated by ethanol, waxed, sliced by a slicer with the size of 3 mu m, dewaxed by dimethylbenzene, H & E staining process, the structural change of the lung tissues is observed under a microscope, and finally pathological result analysis is carried out.
2. The improved novel noninvasive mouse tracheal instillation molding method of claim 1, wherein: the self-made temporary spirometer for mice described in step S1-6 is characterized in that a plunger in a 1mL syringe is taken out, the plunger is discarded, and then 60 mu LPBS is stored at the top of the syringe to form a closed space, so that a temporary spirometer is generated, and the purpose of the temporary spirometer is to ensure that a cannula is inserted into a trachea rather than an esophagus, and the success of operation can be judged after a bleomycin solution is instilled.
3. The improved novel noninvasive mouse tracheal instillation molding method of claim 1, wherein: the self-made mouse hanging fixing plate in the step S1 is made of wood, the fixing plate and the workbench generate isosceles triangles with base angles of 60-80 degrees, an inclined operation interface is arranged, a bottom supporting surface and a side cross section are arranged, and a number 4.0 line is horizontally arranged above the bottom supporting surface and the side cross section.
4. The improved novel noninvasive mouse tracheal instillation molding method of claim 1, wherein: the prepared medical adhesive tape is loosely fixed on the lower part of the chest of the mouse in the step S2-3, the fixed position and the tightness are noted, and the chest movement range of the mouse is ensured.
5. The improved novel noninvasive mouse tracheal instillation molding method of claim 1, wherein: the condition of seeing the trachea in the step S2-7 is that the flashlight irradiates the neck of the mouse in the trachea instillation process, the trachea is distinguished through the action of breathing, the intensity of emitted light can fluctuate through breathing, when the position is correct, the condition is distinguished as a light needle located at the center on the axial plane, and the ambient light of the oral cavity is minimum.
6. The improved novel noninvasive mouse tracheal instillation molding method of claim 1, wherein: in steps S2, S3, the 20G plastic catheter needle is replaced with a 16G plastic catheter needle or a 23G plastic catheter needle.
7. The improved novel noninvasive mouse tracheal instillation molding method of claim 1, wherein: and (3) standing the mice on an operating table for 10min in the step (S3).
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| CN110559102A (en) * | 2019-08-30 | 2019-12-13 | 南华大学 | Rat non-exposure type tracheal instillation method |
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