阅读说明：本技术 大鼠非暴露式气管滴注方法 (Rat non-exposure type tracheal instillation method ) 是由 张朝晖 王烨 雷媛娣 李鹏 谭滇湘 于 2019-08-30 设计创作，主要内容包括：大鼠非暴露式气管滴注方法,步骤如下：1,制作插管；2,麻醉；3,固定；4,气管插管；5,滴注；6,注气；7,复苏。本发明提供的大鼠非暴露式气管滴注方法中,气管插管操作简单易上手,成功率高,对器械和设备的要求较低,无需使用昂贵设备,受试大鼠在气管插管及滴注过程中受伤的概率较小,可广泛应用于呼吸道给药或染毒试验以及各种疾病模型的建立。(The rat non-exposure type tracheal instillation method comprises the following steps: 1, manufacturing a cannula; 2, anaesthetizing; 3, fixing; 4, trachea intubation; 5, instillation; 6, injecting gas; and 7, recovering. In the rat non-exposure type tracheal instillation method provided by the invention, the tracheal intubation operation is simple and easy to operate, the success rate is high, the requirements on instruments and equipment are low, expensive equipment is not required, the probability of injury of the tested rat in the tracheal intubation and instillation process is low, and the rat non-exposure type tracheal instillation method can be widely applied to respiratory tract administration or contamination tests and the establishment of various disease models.)

1. The rat non-exposure type tracheal instillation method is characterized in that: the method comprises the following steps:

S01, manufacturing a cannula: selecting a No. 9 disposable sterile syringe needle, grinding the tip of the No. 9 disposable sterile syringe needle to be round and blunt, bending the needle at a position 10mm away from one end of the needle ground to be round and blunt, wherein the bending angle a is 14-16 degrees, and the needle is named as a rat trachea cannula, one end of the rat trachea cannula is a liquid outlet end, and the other end of the rat trachea cannula is a syringe connecting end;

s02, anesthesia: selecting adult rats, anesthetizing the adult rats by using diethyl ether, and entering the next step by using the adult rats with relaxed muscles, disappearance of eyelid closing reflex and no contraction reaction of pinching skin or toes by using toothed forceps;

s03, fixing: placing the anesthetized rat on an operating table to a supine position, and fixing incisors on the rat on the operating table by rubber bands and four limbs by cotton ropes;

s04, endotracheal intubation: aligning a light source of a transmission flashlight to the lower jaw of a tested rat, wrapping the tip end of a tissue forceps by a medical adhesive tape, slightly pulling the tongue of the tested rat out of the oral cavity towards one side of the corner of the mouth by using the tissue forceps, adjusting the light angle of the transmission flashlight to enable the laryngeal and laryngeal pipelines of the throat of the tested rat to be visible, then wiping off secretion in the throat of the tested rat by using a sterile cotton swab, extending the liquid outlet end of a tracheal cannula of the rat into the oral cavity from the position between the upper incisor and the lower incisor of the tested rat, and slightly pushing the liquid outlet end of the tracheal cannula of the rat towards the fornix of the hard palate along the curve of the oral cavity so as to avoid damaging the oral cavity and the throat of the rat, so that the tracheal cannula of the rat is inserted into the trachea of the rat, wherein the insertion depth is based on the tracheal cannula;

S05, instillation: sucking 0.6-1.2 ml of infusion solution by using a disposable sterile syringe, then connecting the infusion solution to the needle head connecting end of the rat trachea cannula, and slowly injecting the infusion solution at a constant speed so that the infusion solution gradually drops into the rat trachea;

S06, gas injection: after instillation is completed, taking down the disposable sterile syringe, extracting 0.4-0.6 ml of air by using another disposable sterile syringe, then connecting the disposable sterile syringe to the needle head connecting end of the rat trachea cannula, and injecting the air rapidly, wherein the injection time is controlled within 1 second, so that the instillation liquid can be better diffused to the deep part of the trachea;

S07, resuscitation: and (3) pulling out the rat trachea cannula within 5 seconds after the gas injection, holding the tail of the tested rat, hanging the rat for 4-6 seconds, and then putting down the rat until the anesthesia effect disappears.

2. The rat non-exposed tracheal instillation method of claim 1, wherein: in step S01, the bending angle a is 15 °.

3. the rat non-exposed tracheal instillation method of claim 1, wherein: in the step S03, the operating table is tilted 74 to 76 ° with respect to the horizontal plane, and the head of the rat is upward after the rat is fixed.

4. a rat non-exposed tracheal instillation method according to claim 3, wherein: in step S03, the operating table is tilted 75 ° with respect to the horizontal plane.

5. A rat non-exposed tracheal instillation method according to claim 3, wherein: in step S04, the insertion depth is determined by the remaining 25% of the length of the rat' S trachea cannula outside the upper incisors of the rat.

6. The rat non-exposed tracheal instillation method of claim 5, wherein: in step S04, during the process of inserting the rat tracheal cannula into the rat trachea, the thickened respiratory sound is heard through the stethoscope, which indicates the success of the tracheal cannula.

7. The rat non-exposed tracheal instillation method of claim 6, wherein: and S04, in the process of inserting the rat trachea cannula into the rat trachea, if thickened breath sounds are not heard through the stethoscope and the rat trachea cannula is mistakenly inserted into the rat esophagus, performing a second intubation operation, namely, extending the liquid outlet end of the second rat trachea cannula into the oral cavity from the position between the upper incisor and the lower incisor of the tested rat, slightly advancing the liquid outlet end of the second rat trachea cannula along the curve of the oral cavity and towards the fornix of the hard palate, so that the second rat trachea cannula is inserted into the rat trachea, and finally, pulling out the rat trachea cannula mistakenly inserted into the rat esophagus.

8. The rat non-exposed tracheal instillation method of any one of claims 1-7, wherein: in step S05, the amount of the instillation solution was 1.0ml by aspiration with a disposable sterile syringe.

9. The rat non-exposed tracheal instillation method of claim 8, wherein: in step S06, another disposable sterile syringe was used to draw 0.5ml of air.

Technical Field

The invention relates to the field of respiratory system disease experimental research, in particular to a rat non-exposure type tracheal instillation method.

Background

The rat tracheal instillation technology is commonly used for rat tracheal administration, rat tracheal administration modeling, rat lung function determination and experimental research related to respiratory diseases. The trachea instillation is carried out during rat related model replication and rat lung function detection, the trachea instillation technology directly influences the success rate of kinetic model replication and success or failure of lung function detection, and the method is very critical in the whole experimental process.

Tracheal instillation is a method of delivering drugs into the lungs through a tracheal tube, which does not require exposure of the trachea through a surgical incision, and is less harmful to the subject being administered, as compared to administration through a tracheal tube. For larger animals (e.g., rabbits or dogs), the tracheal instillation procedure can be accomplished using a standard pediatric laryngoscope. However, for rats, due to the special anatomical conditions of oral cavity and pharynx, no standard intubation device is available for rats at present, instruments used by other species need to be adjusted generally, professional training is needed to perform manual operation, the actual operation difficulty is high, and the intubation success rate is difficult to guarantee.

At present, the following methods are mainly used for evaluating whether rat trachea intubation is successful: 1. placing a mirror at the front end of the intubation tube, and checking whether fog is formed on the mirror when the rat exhales; 2. connecting the cannula to a rodent lung ventilator and observing whether the thoracic cage of the rat can fluctuate regularly with the mechanical ventilation; 3. dripping a drop of water into an extension tube (a vein indwelling needle hose) and connecting the extension tube with the tail end of the cannula, and observing whether the water in the extension tube synchronously vibrates along with the respiration of the rat; 4. constructing an automatic video drip chamber (AVI) to visually evaluate whether the intubation is successful; 5. a rat cilium is fixed at the intubation port in an adhering mode, and whether the rat cilium swings along with the breathing of the rat is observed.

The above methods have the following disadvantages: in method 1, the mist on the mirror generally dissipates faster, making the method unreliable; in the method 2, the rodent lung breathing machine is expensive, and most laboratories do not have the equipment, so that the method is difficult to popularize; in the method 3, the operation steps are more complicated, and the time required for evaluation is longer compared with other methods; in the method 4, expensive hardware equipment is required for supporting the construction of an automatic video drip chamber (AVI), and corresponding equipment is not available in most laboratories, so that the method is difficult to popularize; in the method 5, the inner diameter of the intubation tube of the rat is small, the ventilation volume of the rat during respiration is small, the swinging amplitude of cilia of the rat is small, and the cilia are difficult to observe by naked eyes, so that the method is unreliable.

in conclusion, the research of the rat trachea instillation method which is simple and convenient to operate, high in success rate and low in requirement on expensive equipment and the rat trachea cannula success or failure evaluation method which is simple and convenient to operate, good in reliability and low in requirement on expensive equipment have very important significance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a rat non-exposure type tracheal instillation method. The device solves the problems that in the existing rat tracheal instillation operation, the difficulty of tracheal intubation is high, the intubation success rate is low, and whether the tracheal intubation is successful or not is difficult to judge.

The technical scheme of the invention is as follows: the rat non-exposure type tracheal instillation method comprises the following steps:

S01, manufacturing a cannula: selecting a No. 9 disposable sterile syringe needle, grinding the tip of the No. 9 disposable sterile syringe needle to be round and blunt, bending the needle at a position 10mm away from one end of the needle ground to be round and blunt, wherein the bending angle a is 14-16 degrees, and the needle is named as a rat trachea cannula, one end of the rat trachea cannula is a liquid outlet end, and the other end of the rat trachea cannula is a syringe connecting end;

S02, anesthesia: selecting adult rats, anesthetizing the adult rats by using diethyl ether, and entering the next step by using the adult rats with relaxed muscles, disappearance of eyelid closing reflex and no contraction reaction of pinching skin or toes by using toothed forceps;

S03, fixing: placing the anesthetized rat on an operating table to a supine position, and fixing incisors on the rat on the operating table by rubber bands and four limbs by cotton ropes;

s04, endotracheal intubation: aligning a light source of a transmission flashlight to the lower jaw of a tested rat, wrapping the tip end of a tissue forceps by a medical adhesive tape, slightly pulling the tongue of the tested rat out of the oral cavity towards one side of the corner of the mouth by using the tissue forceps, adjusting the light angle of the transmission flashlight to enable the laryngeal and laryngeal pipelines of the throat of the tested rat to be visible, then wiping off secretion in the throat of the tested rat by using a sterile cotton swab, extending the liquid outlet end of a tracheal cannula of the rat into the oral cavity from the position between the upper incisor and the lower incisor of the tested rat, and slightly pushing the liquid outlet end of the tracheal cannula of the rat towards the fornix of the hard palate along the curve of the oral cavity so as to avoid damaging the oral cavity and the throat of the rat, so that the tracheal cannula of the rat is inserted into the trachea of the rat, wherein the insertion depth is based on the tracheal cannula;

S05, instillation: sucking 0.6-1.2 ml of infusion solution by using a disposable sterile syringe, then connecting the infusion solution to the needle head connecting end of the rat trachea cannula, and slowly injecting the infusion solution at a constant speed so that the infusion solution gradually drops into the rat trachea;

s06, gas injection: after instillation is completed, taking down the disposable sterile syringe, extracting 0.4-0.6 ml of air by using another disposable sterile syringe, then connecting the disposable sterile syringe to the needle head connecting end of the rat trachea cannula, and injecting the air rapidly, wherein the injection time is controlled within 1 second, so that the instillation liquid can be better diffused to the deep part of the trachea;

S07, resuscitation: and (3) pulling out the rat trachea cannula within 5 seconds after the gas injection, holding the tail of the tested rat, hanging the rat for 4-6 seconds, and then putting down the rat until the anesthesia effect disappears.

the further technical scheme of the invention is as follows: in step S01, the bending angle a is 15 °.

The further technical scheme of the invention is as follows: in the step S03, the operating table is tilted 74 to 76 ° with respect to the horizontal plane, and the head of the rat is upward after the rat is fixed.

the invention further adopts the technical scheme that: in step S03, the operating table is tilted 75 ° with respect to the horizontal plane.

the invention further adopts the technical scheme that: in step S04, the insertion depth is determined by the remaining 25% of the length of the rat' S trachea cannula outside the upper incisors of the rat.

The further technical scheme of the invention is as follows: in step S04, during the process of inserting the rat tracheal cannula into the rat trachea, the thickened respiratory sound is heard through the stethoscope, which indicates the success of the tracheal cannula.

The further technical scheme of the invention is as follows: and S04, in the process of inserting the rat trachea cannula into the rat trachea, if thickened breath sounds are not heard through the stethoscope and the rat trachea cannula is mistakenly inserted into the rat esophagus, performing a second intubation operation, namely, extending the liquid outlet end of the second rat trachea cannula into the oral cavity from the position between the upper incisor and the lower incisor of the tested rat, slightly advancing the liquid outlet end of the second rat trachea cannula along the curve of the oral cavity and towards the fornix of the hard palate, so that the second rat trachea cannula is inserted into the rat trachea, and finally, pulling out the rat trachea cannula mistakenly inserted into the rat esophagus.

the further technical scheme of the invention is as follows: in step S05, the amount of the instillation solution was 1.0ml by aspiration with a disposable sterile syringe.

The further technical scheme of the invention is as follows: in step S06, another disposable sterile syringe was used to draw 0.5ml of air.

Compared with the prior art, the invention has the following advantages:

1. In the rat non-exposure type tracheal instillation method provided by the invention, the tracheal intubation operation is simple and easy to operate, the success rate is high, the requirements on instruments and equipment are low, expensive equipment is not required, the probability of injury of the tested rat in the tracheal intubation and instillation process is low, and the rat non-exposure type tracheal instillation method can be widely applied to respiratory tract administration or contamination tests and the establishment of various disease models.

2. In the rat non-exposure type tracheal instillation method provided by the invention, the method for judging whether the tracheal intubation succeeds is simple, rapid and good in reliability, expensive equipment is not required, and good guarantee is provided for smooth implementation of tracheal instillation.

The invention is further described below with reference to the figures and examples.

Drawings

FIG. 1 is a schematic structural view of a rat trachea cannula;

FIG. 2 is a graph showing the observation result of pathological examination of rat lung tissues in a specific application example of the present invention.

Detailed Description