CN104784682A - Method for noninvasive production of chronic obstructive pulmonary disease acute exacerbation animal model - Google Patents
Method for noninvasive production of chronic obstructive pulmonary disease acute exacerbation animal model Download PDFInfo
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- CN104784682A CN104784682A CN201510039328.9A CN201510039328A CN104784682A CN 104784682 A CN104784682 A CN 104784682A CN 201510039328 A CN201510039328 A CN 201510039328A CN 104784682 A CN104784682 A CN 104784682A
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Abstract
The invention discloses a method for noninvasive production of a chronic obstructive pulmonary disease acute exacerbation animal model, a bacterium solution instilling manner of endoscope-guided intratracheal instillation is used, the equipment are as follows: a nose 0 degree 4 # hard endoscope is used, a xenon lamp cold light source is used, and a panasonic KS-822 videography surveillance system is used; a rat is placed in a dryer with the diameter of 26 cm for ether anesthesia, after satisfied anesthesia, the rat is rapidly taken from an anesthetic machine, and placed in a supine position, limbs of the rat are respectively fixed on a rat board with elastic fixing clamps, 6 u (0.15 ml) of elastase solution (7.7 mu/mg, Sigma company) is slowly instilled, 100 mu l of air is then filled, at the moment, the rat is in inspiratory phase, drugs enter into bronchia along with air flow during inspiration, the rat recovery time is 30-60 seconds, and then the rat is placed in a constant temperature animal room for breeding; after inoculation, the general situations of activity, food taking, spirit of the rat are daily observed, and corresponding data is recorded.
Description
Technical field
The invention belongs to animal experimental model constructing technology field, specifically, relate to a kind of method that noinvasive makes acute exacerbations in patients with chronic obstructive pulmonary disease animal model.
Background technology
The construction method of current acute exacerbations in patients with chronic obstructive pulmonary disease animal model has a lot, and the method for the longest use at present comprises: endotoxin inject through tracheal strips add incense cigarette method, tracheal strips instillation protease adds sulfur dioxide stimulus method, persistent bacterial infection adds in smoking method, air flue to inject and injects protease in short elastoser and lipopolysaccharide, air flue and add normal pressure low oxygen method, CaCl
2associating mountain Semen stizolobii capitati class preparation.Above-mentioned modeling method all needs instillation chemicals in air flue, and because the structure time of acute exacerbations in patients with chronic obstructive pulmonary disease animal model is longer, therefore in modeling process, the survival rate of animal is a very crucial problem.In modeling process, the step of instillation chemicals is quite crucial in air flue, and can have tracheotomy method, direct-view lower dose regimen, blindmate method etc., all belonging to has wound to operate, and causes damage to a certain degree, can affect amount of survival and the quality of molding animal to animal.
Tracheotomy method: for there being wound to operate, simple, and after lancing, the visual field exposes good, is easy to accurate location; But postoperative infection whether and operation the destruction of trachea local and surrounding tissue is difficult to grasp, survival rate is low.
The lower dose regimen of direct-view: after choosing light source exposure glottis, operator is when looking at glottis straight, carry out the associative operation of intrarterial, little compared with additive method wound, apparatus is corresponding with technical merit requirement also to be reduced, but selects the clear exposure glottis of suitable light source to be then this method key link, multiselect bulb, cold light source, torch etc. are through the light of cervical region.Now be aided with the tractive root of the tongue and expose glottis, but rat head is long and narrow, glottis is narrow and small and in deep, oral cavity, when the doser selected can not coordinate physiological camber very well as crossed thick or form, moving closer to epiglottis portion, then can block most of sight line, within the blink that glottis exposes, more difficult accurate insertion glottis complete operation, and easily by doser enter the esophagus by mistake or because the unclear repeated multiple times insertion in the visual field causes laryngeal edema, Airway damage, vocal cords are hemorrhage, even rats death time serious.
Blindmate method: per os intrarterial, when not exposing glottis, easy repetitious stimulation throat, causes myxedema hemorrhage, and airway secretions increases, and cause airway obstruction, very easily injection device is strayed into esophagus in addition, success rate is lower.Percutaneous intrarterial, then need directly accurately to locate position of trachea from the administration of cricoid cartilage gap from external skin, higher to the technical requirement of operator, otherwise easily repeatedly stimulate local muscle and fascia etc., administering effect is difficult to assessment, and also easily cause edema hemorrhage, success rate is low.
For above-mentioned existing operating technology, at present, need a kind of easy to operate, success rate is high, damages rat little and with low cost intrarterial method.
Summary of the invention
In order to overcome the defect existed in prior art, the present invention proposes a kind of method that noinvasive makes acute exacerbations in patients with chronic obstructive pulmonary disease animal model.Its technical scheme is as follows:
Noinvasive makes a method for acute exacerbations in patients with chronic obstructive pulmonary disease animal model, comprises the following steps:
Step 1, bacterial strain and antibacterial culturing
Streptococcus pneumoniae reference culture is provided by Clinical Laboratory center, Tianjin, bacterial strain number: ATCC49619, get strains A TCC49619 and be inoculated in blood agar culture dish, cultivate 18-24h for 37 DEG C, picking list colony inoculation THB fluid medium, 37 DEG C, 200rpm/min shaken cultivation 12h, after centrifugal concentrating, for subsequent use;
Step 2, laboratory animal
Cleaning grade Wistar male rat 60, monthly age 3-6 month, body weight 180 scholar 20g, was provided by Beijing HFK Bio-Technology Co., Ltd., animal qualified (license) card number: SCXK (capital) 2009-0004; Experimental rat is fed in Tianyin Test Animal Centre, and feeding environment indoor relative humidity is 50%-60%, and temperature controls at 18-22 DEG C; Conventional feed is fed, free drinking water, and ventilate on time and wash away feces, keep environment quiet, adaptability starts experiment after feeding 1 week;
Step 3, emphysema animal model build:
Adopt endoscope to guide the instillation bacterium liquid mode of lower tracheal strips perfusion, equipment adopts nose 0 degree of rigid endoscope of 4#, xenon lamp cold light source and Panasonic KS-822 to shoot with video-corder surveillance;
Rat is placed in etherization in exsiccator that diameter is 26cm, rapid after anesthesia is satisfied Mus is taken out from anesthetic machine, with elastic force fixation clamp, its extremity are individually fixed on Mus plate in dorsal position shape;
Two operators are positioned at direction, the Mus crown side by side, first assistant holds Mus anesthetic laryngoscope with left hand, oral cavity is stretched into from the upper lips and teeth of Mus, pressure-raising Mus tongue, the Mus root of the tongue is lifted when extending Mus epiglottis edge place, tractive lasso is held with the right hand, entangle the above-listed tusk front tooth of Mus, right-hand man is the upper and lower tractive root of the tongue of rightabout and tusk front tooth simultaneously, make Mus unimpeded exposure glottis region of upper breathing between lips and teeth to throat, now dominant operator holds 0 degree of rigid endoscope with left hand, the right hand holds No. 7 long-needled syringe, in-built quantitative modeling streptococcus pneumoniae bacteria suspension used, under monitor display guides, No. 7 long entry needles are inserted tracheal strips, and 15 ~ 20mm under going deep into glottis, now observe the vocal cords active situation of Mus, when bilateral vocal cords are changed to abduction shape by adduction shape, slow instillation 6u 0.15ml elastin laminin enzymatic solution 7.7 μ/mg, Sigma company, inject 100 μ l air subsequently, now Mus is expiratory phase, medicine is fully entered in bronchus with air-flow during air-breathing, after injecting, Mus plate is erected rapidly Mus head upper left and right upset 15 ~ 20 seconds, blank group does not process, saline control group injects the physiological saline solution of isodose, within every 7 days, instill once, totally 3 times, 21 days modeling time,
Mus is put back in mouse cage and waits to recover by solution deratization extremity elastic force fixation clamp, about 30 ~ 60 seconds Mus recovery time, is then placed in Homoiotherm room and raises; After inoculation every day observe that it is movable, take food, the ordinary circumstance of spirit, record corresponding data;
Step 4, preparation streptococcus pneumoniae agar beads suspension:
Streptococcus pneumoniae reference culture is provided by Clinical Laboratory center, Tianjin, bacterial strain number: ATCC49619; After nutrient agar panel is cultivated, adjustment bacterial concentration is 2 × 10
12-6 × 10
12cFU/ml, gets concentrated bacterium liquid and 45 DEG C of agar solution mixed in equal amounts of dissolving, and injects rapidly the brain-heart infusion medium of 4 DEG C of rapid stirrings, makes the streptococcus pneumoniae agar beads suspension being wrapped up antibacterial by agar; It is 1 × 10 that suspension comprises bacterial concentration
12-3 × 10
12the unsetting agar beads of CFU/ml.
Beneficial effect of the present invention is:
The method that the present invention uses noinvasive means tracheal strips to instill elastoser and streptococcus pneumoniae builds chronic obstructive pulmonary disease animal model.The method, by noninvasive method, decreases the damage to animal, improves survival rate, ensure that animal can avoid having wound to operate the death brought in the modeling process of long period.Research finds that the elastoser that at every turn can ensure to instill q.s can shorten the modeling time relatively, also saves modeling cost while saving the time.
Accompanying drawing explanation
Fig. 1 is that naive animals lung tissue HE dyes (50 times);
Fig. 2 is that model group animal lung tissue HE dyes (50 times);
Fig. 3 is that saline control treated animal lung tissue HE dyes (50 times).
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in more detail.A kind of noinvasive makes the method for acute exacerbations in patients with chronic obstructive pulmonary disease animal model
Step 1, bacterial strain and antibacterial culturing
Streptococcus pneumoniae reference culture is provided by Clinical Laboratory center, Tianjin, bacterial strain number: ATCC49619, get strains A TCC49619 and be inoculated in blood agar culture dish, cultivate 18-24h for 37 DEG C, picking list colony inoculation THB fluid medium, 37 DEG C, 200rpm/min shaken cultivation 12h, after centrifugal concentrating, for subsequent use.
Step 2, laboratory animal
Cleaning grade Wistar male rat 60, monthly age 3-6 month, body weight 180 scholar 20g, was provided by Beijing HFK Bio-Technology Co., Ltd., animal qualified (license) card number: SCXK (capital) 2009-0004; Experimental rat is fed in Tianyin Test Animal Centre, and feeding environment indoor relative humidity is 50%-60%, and temperature controls at 18-22 DEG C; Conventional feed is fed, free drinking water, and ventilate on time and wash away feces, keep environment quiet, adaptability starts experiment after feeding 1 week.
Step 3, emphysema animal model build:
Adopt endoscope to guide the instillation bacterium liquid mode of lower tracheal strips perfusion, equipment adopts nose 0 degree of rigid endoscope of 4#, xenon lamp cold light source and Panasonic KS-822 to shoot with video-corder surveillance;
Rat is placed in etherization in exsiccator that diameter is 26cm, rapid after anesthesia is satisfied Mus is taken out from anesthetic machine, with elastic force fixation clamp, its extremity are individually fixed on Mus plate in dorsal position shape;
Two operators are positioned at direction, the Mus crown side by side, first assistant holds Mus anesthetic laryngoscope with left hand, oral cavity is stretched into from the upper lips and teeth of Mus, pressure-raising Mus tongue, the Mus root of the tongue is lifted when extending Mus epiglottis edge place, tractive lasso is held with the right hand, entangle the above-listed tusk front tooth of Mus, right-hand man is the upper and lower tractive root of the tongue of rightabout and tusk front tooth simultaneously, make Mus unimpeded exposure glottis region of upper breathing between lips and teeth to throat, now dominant operator holds 0 degree of rigid endoscope with left hand, the right hand holds No. 7 long-needled syringe, in-built quantitative modeling streptococcus pneumoniae bacteria suspension used, under monitor display guides, No. 7 long entry needles are inserted tracheal strips, and 15 ~ 20mm under going deep into glottis, now observe the vocal cords active situation of Mus, when bilateral vocal cords are changed to abduction shape by adduction shape, slow instillation 6u (0.15ml) elastin laminin enzymatic solution (7.7 μ/mg, Sigma company), inject 100 μ l air subsequently, now Mus is expiratory phase, medicine is fully entered in bronchus with air-flow during air-breathing, after injecting, Mus plate is erected rapidly Mus head upper left and right upset 15 ~ 20 seconds, blank group does not process, saline control group injects the physiological saline solution of isodose, within every 7 days, instill once, totally 3 times, 21 days modeling time.
Mus is put back in mouse cage and waits to recover by solution deratization extremity elastic force fixation clamp, about 30 ~ 60 seconds Mus recovery time, is then placed in Homoiotherm room and raises; After inoculation every day observe that it is movable, take food, the ordinary circumstance of spirit, record corresponding data
Step 4, preparation streptococcus pneumoniae agar beads suspension:
Streptococcus pneumoniae reference culture is provided by Clinical Laboratory center, Tianjin, bacterial strain number: ATCC49619.After nutrient agar panel is cultivated, adjustment bacterial concentration is 2 × 10
12-6 × 10
12cFU/ml, gets concentrated bacterium liquid and 45 DEG C of agar solution mixed in equal amounts of dissolving, and injects rapidly the brain-heart infusion medium of 4 DEG C of rapid stirrings, makes the streptococcus pneumoniae agar beads suspension being wrapped up antibacterial by agar.It is 1 × 10 that suspension comprises bacterial concentration
12-3 × 10
12the unsetting agar beads of CFU/ml.
The foundation of acute exacerbations in patients with chronic obstructive pulmonary disease animal model:
After emphysema animal model causes, S. pneumoniae challenge is used to build AECOPD model.(still adopt endoscope to guide the instillation bacterium liquid mode of lower tracheal strips perfusion, equipment adopts nose 0 degree of rigid endoscope of 4#, xenon lamp cold light source and Panasonic KS-822 to shoot with video-corder surveillance)
Rat is placed in etherization in exsiccator that diameter is 26cm, rapid after anesthesia is satisfied Mus is taken out from anesthetic machine, with elastic force fixation clamp, its extremity are individually fixed on Mus plate in dorsal position shape;
Two operators are positioned at direction, the Mus crown side by side, first assistant holds Mus anesthetic laryngoscope with left hand, oral cavity is stretched into from the upper lips and teeth of Mus, pressure-raising Mus tongue, the Mus root of the tongue is lifted when extending Mus epiglottis edge place, tractive lasso is held with the right hand, entangle the above-listed tusk front tooth of Mus, right-hand man is the upper and lower tractive root of the tongue of rightabout and tusk front tooth simultaneously, make Mus unimpeded exposure glottis region of upper breathing between lips and teeth to throat, now dominant operator holds 0 degree of rigid endoscope with left hand, the right hand holds No. 7 long-needled syringe, in-built quantitative modeling streptococcus pneumoniae bacteria suspension used, under monitor display guides, No. 7 long entry needles are inserted tracheal strips, and 15 ~ 20mm under going deep into glottis, now observe the vocal cords active situation of Mus, when bilateral vocal cords are changed to abduction shape by adduction shape, (bacterial concentration is 1 × 10 to slow instillation streptococcus pneumoniae agar suspension 0.15ml
12cFU/mL), and inject a small amount of air to ensure that whole antibacterial enters trachea, upright for animal also appropriateness is shaken 15-20 second, makes antibacterial be uniformly distributed in two lungs as far as possible.Place rat to lie low to reviving.Be placed in 18-22 DEG C of Homoiotherm room to continue to raise.
Experimental result
Animal ordinary circumstance
Animal inoculation elastoser the 1st day, dead 2, be anesthesia procedure error and lethal, have nothing to do with modeling method, complete supplementary subsequently, negative control group rat is vivaciously active, strong big and fleshy, gloss, breathes steadily; Modeling treated animal is movable gradually to be reduced, and roll up asthenia, hogback erects hair, and hair tarnishes, and breathes frequently short, time have sneeze, after 3 hours, activity increases, but spirit is slightly poor.Inoculate in 24 hours in the animal of streptococcus pneumoniae, matched group does not have death, and animal pattern infected group has 1 animal dead, and take food all as seen minimizing, energy of all the other animals declines, and after 3 days, above-mentioned symptom obviously alleviates, and operation is drawn materials before day and do not occurred death again.
Animal generally appearance activity minimizing in 3 days after note bacterium, cough symptom is obvious, and model group is also with comparatively significantly panting; After one week, intact animal's infected group is several asymptomatic, and substantially return to normal, animal pattern infected group still can see rapid breathing after three weeks, occasionally has cough.
Animal lung tissue's pathology
Gross examination of skeletal muscle: model group lung volume compared with normal matched group obviously increases, color is pale, the fusion of pulmonary alveoli that surface has quantity not wait is formed excessively inflates bulla, elastance of lung weakens, cut open breast after the not atrophy of lung stolen goods, still keep swelling state, compared with model group, normal rats lung tissue changes not obvious substantially.
With reference to Fig. 1-Fig. 3, light Microscopic observation:
Blank group (Fig. 1): naked eyes are shown in that lung volume is normal, and elasticity is normal; Under mirror, bronchial mucosa epithelial structure is complete, bronchus cilium marshalling, alveolar marshalling, structural integrity, and without significantly destroying, alveolar wall is without obviously thickening.Have no cell infiltration.
Model group (Fig. 2): under mirror, visible bronchial epithelial cell portion deforms is downright bad, partial exfoliation, alveolar differs in size, and alveolar structure is disorderly, and alveolar space increases, alveolar wall is thinning, and having fracture in various degree, partial fusion becomes pulmonary belb, has more visible massive inflammatory cells infiltrated around, the visible tube wall thickening of lung small artery, massive inflammatory cells infiltrated around tube wall.
Saline control group (Fig. 3): alveolar marshalling under mirror, structural integrity, without significantly destroying, alveolar wall is without obviously thickening.The visible mild inflammatory cellular infiltration of surrounding alveolar walls.
The above; be only the present invention's preferably detailed description of the invention; protection scope of the present invention is not limited thereto; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the simple change of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (2)
1. noinvasive makes a method for acute exacerbations in patients with chronic obstructive pulmonary disease animal model, it is characterized in that, comprises the following steps:
Step 1, bacterial strain and antibacterial culturing
Streptococcus pneumoniae reference culture is provided by Clinical Laboratory center, Tianjin, bacterial strain number: ATCC49619, get strains A TCC49619 and be inoculated in blood agar culture dish, cultivate 18-24h for 37 DEG C, picking list colony inoculation THB fluid medium, 37 DEG C, 200rpm/min shaken cultivation 12h, after centrifugal concentrating, for subsequent use;
Step 2, laboratory animal
Cleaning grade Wistar male rat 60, monthly age 3-6 month, body weight 180 scholar 20g, was provided by Beijing HFK Bio-Technology Co., Ltd., the qualified credit number of animal: SCXK (capital) 2009-0004; Experimental rat is fed in Tianyin Test Animal Centre, and feeding environment indoor relative humidity is 50%-60%, and temperature controls at 18-22 DEG C; Conventional feed is fed, free drinking water, and ventilate on time and wash away feces, keep environment quiet, adaptability starts experiment after feeding 1 week;
Step 3, emphysema animal model build:
Adopt endoscope to guide the instillation bacterium liquid mode of lower tracheal strips perfusion, equipment adopts nose 0 degree of rigid endoscope of 4#, xenon lamp cold light source and Panasonic KS-822 to shoot with video-corder surveillance;
Rat is placed in etherization in exsiccator that diameter is 26cm, rapid after anesthesia is satisfied Mus is taken out from anesthetic machine, with elastic force fixation clamp, its extremity are individually fixed on Mus plate in dorsal position shape;
Two operators are positioned at direction, the Mus crown side by side, first assistant holds Mus anesthetic laryngoscope with left hand, oral cavity is stretched into from the upper lips and teeth of Mus, pressure-raising Mus tongue, the Mus root of the tongue is lifted when extending Mus epiglottis edge place, tractive lasso is held with the right hand, entangle the above-listed tusk front tooth of Mus, right-hand man is the upper and lower tractive root of the tongue of rightabout and tusk front tooth simultaneously, make Mus unimpeded exposure glottis region of upper breathing between lips and teeth to throat, now dominant operator holds 0 degree of rigid endoscope with left hand, the right hand holds No. 7 long-needled syringe, in-built quantitative modeling streptococcus pneumoniae bacteria suspension used, under monitor display guides, No. 7 long entry needles are inserted tracheal strips, and 15 ~ 20mm under going deep into glottis, now observe the vocal cords active situation of Mus, when bilateral vocal cords are changed to abduction shape by adduction shape, slow instillation 6u 0.15ml elastin laminin enzymatic solution 7.7 μ/mg, Sigma company, inject 100 μ l air subsequently, now Mus is expiratory phase, medicine is fully entered in bronchus with air-flow during air-breathing, after injecting, Mus plate is erected rapidly Mus head upper left and right upset 15 ~ 20 seconds, blank group does not process, saline control group injects the physiological saline solution of isodose, within every 7 days, instill once, totally 3 times, 21 days modeling time,
Mus is put back in mouse cage and waits to recover by solution deratization extremity elastic force fixation clamp, 30 ~ 60 seconds Mus recovery time, is then placed in Homoiotherm room and raises; After inoculation every day observe that it is movable, take food, the ordinary circumstance of spirit, record corresponding data;
Step 4, preparation streptococcus pneumoniae agar beads suspension:
Streptococcus pneumoniae reference culture is provided by Clinical Laboratory center, Tianjin, bacterial strain number: ATCC49619; After nutrient agar panel is cultivated, adjustment bacterial concentration is 2 × 10
12-6 × 10
12cFU/ml, gets concentrated bacterium liquid and 45 DEG C of agar solution mixed in equal amounts of dissolving, and injects rapidly the brain-heart infusion medium of 4 DEG C of rapid stirrings, makes the streptococcus pneumoniae agar beads suspension being wrapped up antibacterial by agar.
2. noinvasive according to claim 1 makes the method for acute exacerbations in patients with chronic obstructive pulmonary disease animal model, and it is characterized in that, it is 1 × 10 that streptococcus pneumoniae agar beads suspension comprises bacterial concentration
12-3 × 10
12the unsetting agar beads of CFU/ml.
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| CN108570418A (en) * | 2018-04-08 | 2018-09-25 | 山东农业大学 | A kind of aspergillus fumigatus agar beads and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108570418A (en) * | 2018-04-08 | 2018-09-25 | 山东农业大学 | A kind of aspergillus fumigatus agar beads and preparation method thereof |
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