CN114788508A - Method for constructing sudden deafness mouse model by using inflammation as characteristic - Google Patents
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to a method for constructing a sudden deafness mouse model by taking inflammation as a characteristic, which comprises the following steps: 1) adopting a C57BL/6 mouse, punching a hole at the position below the mouse after the mouse cavum and adjacent to the temporal bone, exposing the round window, injecting collagenase into the round window niche of the mouse for infiltration, and absorbing and removing residual collagenase in the round window niche by using gelatin sponge after infiltration; 2) taking 3 mul of LPS solution with the concentration of 5mg/ml, firstly injecting 1 mul of LPS solution into the round window niche, filling the round window niche with gelatin sponge, then injecting the rest 2 mul of LPS solution into the gelatin sponge, suturing muscles and skin layer by layer after injection is finished, and putting the mouse into a cage for continuous feeding after the mouse is awake; 3) detecting the reaction threshold of the auditory brainstem and observing the morphology of the cochlea on the 3 rd day after the operation. The invention improves the permeability of LPS to the periauricular lymph fluid through the round window membrane, avoids the middle ear cavity infection caused by the tympanum puncture method and meets the requirements of research and development.
Description
Technical Field
The invention relates to a method for constructing a mouse model, in particular to a method for constructing a sudden deafness mouse model by taking inflammation as a characteristic.
Background
According to the records of diagnosis and treatment guidelines for sudden deafness (2015) (2015, 50 th of J. Onchioropharynx and neck surgery, Inc.) for sudden deafness, sudden deafness refers to sudden and unexplained sensorineural hearing loss, hearing loss of at least 2 connected frequencies within 3 days is reduced by more than 20dB, the pathogenesis is unclear, the etiology is unclear, the inducement is more, and no satisfactory animal model is available for relevant research The defects of damage to brain tissues and organs of animals and short survival time of the animals are caused. In 2004, Hoya N et al reported that the delivery of the mitochondrial toxin 3-nitropropionic acid (3-NP) to the inner ear via the round window membrane resulted in an acute mitochondrial dysfunction rat model (A novel animal model of the enzyme cochlear mitochondrial dysfunction. Hoya N. neuroreport.2004Jul 19; 15 (10): 1597-. In 2007, 3-NP is respectively reported to be put into a round window membrane to construct a sudden deafness guinea pig model, and the effect of oxidative stress in the model is discussed (establishment of a sudden deafness model with vertigo induced by mitochondrial toxin, Zhang ya, Yanjing, Ilmaripykko, Wuhao, Shanghai university of transportation (medical edition) in 2007, No. 1 of the theory of sudden deafness model induced by mitochondrial toxin, research on the mechanism of vascular streak injury in the same model, Rong, Kouyoujia, Chengmeng, China journal of histochemistry and cytochemistry, 2009, No. 18, No. 2). In 2005, the authors reported that Lipopolysaccharide (LPS) was injected into the middle ear cavity by tympanocentesis, and then permeated into the inner ear through the round window membrane to construct a guinea pig model with inflammatory conditions for animals with sensorineural deafness, which was simple to operate, but resulted in otitis media and affected the accuracy of hearing tests (establishment and evaluation of animal models with infectious neurological deafness, wangnan, wang dynasty, shang chang you, chinese clinical rehabilitation 2005, volume 9, stage 4).
Disclosure of Invention
The applicant found that more and more evidence suggests that the development of sudden deafness is closely related to the inflammatory response. Based on the above findings, the applicant has proposed, through a large number of experiments and studies, a method for constructing a mouse model of acute sensorineural deafness (sudden deafness) characterized by inflammatory responses by a technique of infiltrating a round window membrane with collagenase, then administering LPS, and infiltrating into the inner ear through a round window membrane route. The method comprises the following specific steps:
1. adopting a C57BL/6 mouse, punching a hole at the position below the mouse after the mouse cavum and adjacent to the temporal bone, exposing the round window, injecting collagenase into the round window niche of the mouse for infiltration, and absorbing and removing residual collagenase in the round window niche by using gelatin sponge after infiltration;
2) taking 3 mul of Lipopolysaccharide (LPS) solution with the concentration of 5mg/ml, firstly injecting 1 mul of LPS solution into the round window niche, filling the round window niche with gelatin sponge, then injecting the rest 2 mul of LPS solution into the gelatin sponge, suturing muscles and skin layer by layer after injection is finished, and putting the mouse into a cage for continuous breeding after the mouse is revived;
3. auditory Brainstem Response (ABR) and cochlear morphology were examined on day 3 post-operatively. The average ABR response threshold value is reduced by more than 15dBHL, the cochlear morphology is subjected to inflammation change, and hair cells are deleted, so that the success of the LPS-induced sudden deafness mouse model is shown, and the molecular mechanism of acute injuries of cochlear hair cells, spiral ganglia and the like caused by inflammatory factors in the body state can be researched.
In the step 1), the C57BL/6 mice are male and female, the week age is preferably 6-8 weeks, the bodies of the mice with small ages are relatively poor, the tolerance to surgery and anesthesia is poor, and the average hearing level of the mice with large ages is reduced.
In the step 1), firstly, a 7 # sharp knife is used for expanding the punching range to about 1.5mm in diameter, a stapes artery which is clung to the inner side wall of the tympanic cavity and longitudinally runs can be seen, an oval bony depression is seen at the back upper part of the stapes artery, namely the position of the round window niche, and then microscopic forceps are used for separating tissues around the round window niche, so that the round window is fully exposed.
In step 1), 1. mu.l of collagenase I with a concentration of 30mg/ml was extracted by a micropipette and injected into the mouse round window niche by a microinjection pump.
The applicant finds that in the step 1), too long soaking time of collagenase I in a mouse round window niche can cause tissue structure damage after the round window membrane is over digested, and too short soaking time cannot play a role, and the soaking time is determined to be 10 minutes on the basis of a large number of experiments.
In the step 2), the LPS solution is injected at the speed of 50nl/s so as to avoid overflow of the LPS solution caused by too high speed; the gelatin sponge is filled into the hole of the acoustic bubble, and filled with round window niche with volume of 5-10 mm 3 The volume is suitable.
Compared with the prior art, the invention has the following advantages and progresses:
compared with the prior sudden deafness model, the invention adopts a mouse auditory bulb to open a small window, uses collagenase I to digest histiocyte on the surface of a round window membrane in advance before placing LPS, does not damage epithelial tissues of the round window membrane, and improves the penetration rate of the LPS to the perilymph fluid of the inner ear through the round window membrane.
2, LPS is directly infiltrated on the sponge of the window niche and then infiltrated into the inner ear through the window membrane, so that the middle ear cavity infection caused by a tympanocentesis method is avoided.
3, the sudden deafness models in the past are all constructed by guinea pigs or rats, because the genes of mice and people have extremely high similarity, more and more deafness researches in recent years adopt the gene mice, and the invention adopts C57BL/6 mice to construct the models, thereby meeting the requirements of research and development.
4, because the pathogenesis of sudden deafness is unclear, the pathogenesis is mainly learned to be inner ear blood supply disorder, virus infection, immunologic dysfunction, stress and the like, and sudden deafness caused by all factors has inflammatory reaction.
Drawings
Fig. 1 is a photograph of cochlear HE light microscope of a control group [ Artificial lymph (AP) group ] according to the present invention.
Wherein, fig. 1A is a cochlea HE stained panoramic optical lens photograph, fig. 1B is a cochlea basilar membrane optical lens photograph, fig. 1C is a cochlear blood vessel striation optical lens photograph, and fig. 1D is a cochlea modiolus optical lens photograph.
Fig. 1A and 1B show: the hair cells inside and outside the cochlea are regularly arranged, no hair cells are deleted, the basement membrane is complete, and the spiral ligament is uniformly dyed.
FIG. 1C shows: the cochlear veins are not congested, swollen, ruptured.
Fig. 1D shows: spiral nerve cells in the cochlear modiolus are uniformly dyed and arranged in order without loss, and the plasma-nuclear ratio is uniform.
In the control group, AP is infiltrated into the mouse cochlea round window niche sponge, and the change condition of the cochlea structure is observed through HE staining, and fig. 1A-1D show that the cochlea injury can not be caused by the infiltration technology of the mouse cochlea round window niche sponge.
FIG. 2 is a photograph of an HE staining microscope of LPS group of example (experimental group) of the present invention.
Wherein, fig. 2A is a cochlear panoramic speculum photograph, fig. 2B is a cochlear basilar membrane speculum photograph, fig. 2C is an cochlear angiostriation speculum photograph, and fig. 2D is a cochlear modiolar speculum photograph.
Fig. 2A, 3B show: the spiral ligament structure of the cochlea is loose, and the distance between the spiral ligament structure and the volute is increased.
Fig. 2C shows: the cochlear veins become thin and have a fracture phenomenon.
Fig. 2D shows: the spiral nerve cell vacuole in the cochlear modium is degenerated, and the plasma-nuclear ratio is obviously reduced.
The experimental group adopts the steps of the invention to put LPS into the round window niche of the mouse, and the change condition of the cochlea structure is observed through HE staining. Fig. 1A-1D show that LPS injection through the round window membrane can cause cochlear structure damage.
FIG. 3 is an immunofluorescent staining photomicrograph of cochlear basement membrane plating in a mouse model of sudden deafness.
Of these, fig. 3A is a normal control group showing intact cochlear basement membrane outer hair cells, and fig. 3B is an LPS-administered (molded) group showing a marked absence of outer hair cells (arrow-marked).
FIG. 4 is a graph comparing ABR thresholds of frequencies in LPS-induced sudden deafness mouse model.
LPS group compared to AP group, p < 0.01.
Detailed Description
In order to clearly explain the technical features of the present invention, the present invention will be explained in detail by the following embodiments with reference to the attached drawings.
Construction of sudden deafness mouse model
1. Experimental group (LPS group)
The method and the material are as follows:
1) 10 male C57BL/6 mice 7 weeks old are taken, holes are respectively drilled at the lower part behind the auditory blister and the position close to the temporal bone, a No. 7 sharp knife is used for expanding the drilling range to about 1.5mm in diameter, the longitudinal stapedial artery close to the inner side wall of the tympanic cavity is seen, an oval bony depression is seen at the rear upper part of the stapedial artery, namely the position of the round window niche, micro-tweezers are used for separating the surrounding tissues of the round window niche, after the round window is fully exposed, a micropipette is used for extracting 1 mu l of collagenase I with the concentration of 30mg/ml and injecting the collagenase I into the round window niche of the mice through a microinjection pump, and after 10 minutes of infiltration, the residual collagenase in the round window niche is absorbed by gelatin sponge.
2) Mu.l of an LPS solution having a concentration of 5mg/ml was withdrawn with a micropipette, 1. mu.l of the LPS solution was first injected into the round window niche at a rate of 50nl/s by means of a microinjection pump, and then a volume of about 5 to 10mm was placed in the round window niche 3 The gelatin sponge is filled to the opening of the auditory bulb and is filled with the round window niche, then the remaining 2 mul of LPS solution is injected into the gelatin sponge by a micro-injection pump at the speed of 50nl/s, after the injection is finished, the muscle and the skin are sutured layer by layer, and the mouse is put into a cage for continuous feeding after waking up.
3) On the third day after operation, ABR response threshold detection and cochlear HE staining morphology observation show that the average threshold of each frequency is reduced by more than 15dbHL, and the cochlear morphological structure is subjected to inflammation change, which indicates that the acute inner ear injury model induced by LPS is successfully modeled, and can be used for researching the molecular mechanism of the acute injury of cochlear hair cells caused by inflammatory factors in the body state.
2. Control group (AP group) experiment
1) 10 male 7-week-old C57BL/6 mice were punched at the lower part of the rear of the tympanum and the vicinity of the temporal bone, the hole was enlarged to about 1.5mm in diameter by a No. 7 sharp knife, the longitudinal stapedial artery adhered to the inner side wall of the tympanic cavity was observed, an oval bony depression was observed at the upper and rear parts of the stapedial artery, which was the position of the round window niche, the tissue around the round window niche was separated by a pair of micro-forceps, after the round window was fully exposed, 1 μ l of collagenase I with the concentration of 30mg/ml was extracted by a micropipette, and the collagenase I was injected into the round window niche of the mice by a micro-injection pump, and after 10 minutes of infiltration, the residual collagenase in the round window niche was removed by gelatin sponge.
2) 3. mu.l of AP solution having a concentration of 5mg/ml was taken out by a micropipette, 1. mu.l of AP solution was injected into the round window niche at a rate of 50nl/s by a microinjection pump, and then a volume of about 5 to 10mm was placed in the round window niche 3 The gelatin sponge is filled to the opening of the auditory bulb and is filled with the round window niche, then the remaining 2 mul of LPS solution is injected into the gelatin sponge by a micro-injection pump at the speed of 50nl/s, after the injection is finished, the muscle and the skin are sutured layer by layer, and the mouse is put into a cage for continuous feeding after waking up.
3) And (3) observing the hearing level and cochlear morphological structure of the mouse by ABR detection on the third postoperative day, wherein the average hearing threshold of each frequency is reduced to within 5dBHL, and the cochlear morphological structure is basically normal, which indicates that the cochlear injury cannot be caused by the technical operation of placing collagenase into the round window niche of the cochlear of the mouse and infiltrating the sponge with AP.
3. Cochlear morphological structure observation
AP group: referring to fig. 1A-1D, staining of AP group cochlear HE showed that the inner and outer hair cells were aligned, no hair cell loss, intact basement membrane, and uniformly stained spiral ligament. The blood vessel lines were not congested, swollen, and ruptured. The spiral nerve cells in the modiolus are uniformly dyed and arranged in order without loss, and the plasma-nuclear ratio is uniform.
LPS group: referring to fig. 2A and 2D, the cochlear HE of LPS group is stained with loose structure of spiral ligament and increased distance from the volute. The blood vessel veins become thin, the phenomenon of rupture exists, the vacuole of the spiral nerve cells in the modiolus is degenerated, and the plasma-nuclear ratio is obviously reduced. The changes show that the cochlear morphological structure of the LPS group is obviously changed, which indicates that the modeling method can influence the inner ear of the mouse, and the change of the cochlear morphological structure indicates that the cochlear morphological structure can effectively construct a sudden deafness mouse model.
All mice survived well after operation, and vestibular dysfunction such as unstable gait is not seen. When the cochlea is used for material taking after operation, the punching hole of the auditory bulb is wrapped by normal mucous membrane tissue, the middle ear cavity is clean, and the mucous membrane has no swelling, congestion and effusion.
4. And (3) ABR detection:
LPS group: before the mouse sudden deafness model is constructed, the average ABRIII wave reaction threshold value of each frequency is 39.25 +/-3.01 dBHL, the average ABR threshold value of each frequency of the lateral ear measured on the 3 rd day after operation is 61.25 +/-4.05 dBHL, the difference is obviously increased compared with that before the operation, the difference has statistical significance (P is less than 0.05), the ABR threshold values after the operation at 4kHz, 8kHz, 16kHz and 32kHz are obviously changed compared with that before the operation, and the difference has statistical significance (P is less than 0.05).
AP group: before operation, the average ABRIII wave reaction threshold value of each frequency of the mouse is 38.25 +/-2.877 dBHL, the average ABR threshold value of each frequency of the lateral ear measured on the 3 rd day after operation is 40.00 +/-3.85 dBHL, the difference has no statistical significance (P is larger than 0.05), the ABR threshold values after operation at 4kHz, 8kHz, 16kHz and 32kHz have no significant change compared with the ABR threshold values before operation, and the difference has no statistical significance (P is larger than 0.05).
The results of ABR threshold values of each frequency after surgery of two groups of mice suggest that the ABR threshold values of each frequency of LPS group are obviously increased (see FIG. 4).
5. Cochlear hair cell deletion condition observation by basement membrane slide immunofluorescence staining
FIG. 3 is immunofluorescence staining chart of cochlear basement membrane of mouse model of sudden deafness
Referring to fig. 3, fig. 3A shows the integrity of the arrangement of hair cells on the basal membrane of the AP group cochlear; FIG. 3B shows that in the LPS administration (molding) group, there was a significant loss of outer hair cells (white arrows).
According to the results of cochlear morphological structure observation, ABR detection and observation of cochlear hair cell deletion by basement membrane slide immunofluorescence staining, the method for delivering LPS through a round window membrane path and permeating the LPS into the inner ear is shown to effectively construct a sudden deafness mouse model with inflammation as a characteristic.
Claims (6)
1. A method for constructing a sudden deafness mouse model with inflammation as a characteristic, which is characterized by comprising the following steps:
1) adopting a C57BL/6 mouse, punching a hole at the position below the mouse after the mouse cavum and adjacent to the temporal bone, exposing the round window, injecting collagenase into the round window niche of the mouse for infiltration, and absorbing and removing residual collagenase in the round window niche by using gelatin sponge after infiltration;
2) taking 3 mul of Lipopolysaccharide (LPS) solution with the concentration of 5mg/ml, firstly injecting 1 mul of LPS solution into the round window niche, filling the round window niche with gelatin sponge, then injecting the rest 2 mul of LPS solution into the gelatin sponge, suturing muscles and skin layer by layer after injection is finished, and putting the mouse into a cage for continuous breeding after the mouse is revived;
3) detecting an auditory brainstem response threshold value and observing cochlear morphology on the 3 rd day after operation: the average hearing threshold of each frequency is reduced by more than 15dBHL, the cochlear morphology is subjected to inflammation change, and hair cells are deleted, which shows that the LPS-induced sudden deafness mouse model is successful.
2. The method of claim 1, wherein in step 1), the C57BL/6 mice are male and female, and the week old is 6-8 weeks.
3. The method of claim 1, wherein in step 1), the perforation is enlarged by a No. 7 sharp knife to a diameter of about 1.5mm, and the longitudinal stapedial artery close to the inner side wall of the tympanic cavity is visible, and an oval bony depression is visible at the upper and rear part of the stapedial artery, i.e. the position of the round window niche, and then the tissue around the round window niche is separated by the micro-forceps, so that the round window is fully exposed.
4. The method of claim 1, wherein in step 1), 1 μ l of collagenase I with concentration of 30mg/ml is extracted by a micropipette and injected into the round window niche of the mouse by a micro syringe pump.
5. The method of claim 1, wherein collagenase I is infiltrated in the round window niche of the mouse in step 1) for 10 minutes.
6. The method for constructing a mouse model of sudden deafness characterized by inflammation according to claim 1, wherein in step 2), the LPS solution is injected at a rate of 50nl/s, and gelfoam is filled into the alveolar hole, filling the round window niche, and the volume is 5-10 mm 3 。
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