JP6532034B2 - Method of measuring nasal resistance, method of measuring nasal resistance and lung airway resistance, and measuring device used for carrying out the method - Google Patents

Description

  The present invention relates to a measurement method and a measurement apparatus for evaluating upper and lower airway hyperresponsiveness in a mouse model of airway inflammation disease such as allergic rhinitis and asthma.

  At present, an evaluation method using airway resistance and compliance of the lower airway (lung) due to methacholine load is being used as an evaluation of airway hyperresponsiveness in an asthma mouse model, and the evaluation is a mechanism elucidation of asthma and a new treatment It is an essential way to establish the law.

  For example, in Non-Patent Document 1, a respiratory resistance measuring apparatus by oscillation method is prepared for mice, and mice sensitized to ovalbumin (ovalbumin: hereinafter referred to as OVA) and non-sensitized mice are loaded with methacholine. The results of examining the airway hypersensitivity have been shown.

  Also, in Non-Patent Documents 2 and 3, a tube is set toward the upper airway while ventilating the lungs, and the OVA is nasally administered to an allergic rhinitis mouse model against OVA, and nasal resistance 24 hours later (nasal The result of measuring airway resistance (hereinafter referred to as Rna) is disclosed.

  Generally, there are noninvasive whole body plethysmorgraphy (hereinafter referred to as WPG) method and invasive invasive plethysmorgraphy (hereinafter referred to as IPG) method of tracheostomy and tracheostomy to measure airway resistance. . However, in recent years, the latter method has become mainstream due to the reliability of the data, and evaluation of the experimental results by the WBP method is in a situation where it is difficult to be received by the top journals. Currently, as measuring instruments of IPG method, Scireq Flexi Vent system sold by EMKA of France and Buxco Fine Pointe system sold by DSI of USA are sold, and many products are academia and pharmaceuticals. Sold to companies.

Suko Matsunobu et al., Allergy 39 (12), 1629-1632 (1990) Miyahara S. et al, JACI 2005; 116: 1020-7 Miyahara Atsuko et al, The Japanese Pharmacology magazine 2011; 137: 141-145

  The respiratory resistance measurement device described in Non-Patent Document 1 can only evaluate the lower respiratory tract (lung airway), and in the elucidation of the mechanism of allergic rhinitis and drug development, the measurement of nasal resistance at the animal experiment level is established. At present, it is at present substituted by measurement results based on airway resistance in the lower airway.

  Non-Patent Document 2 discloses a method in which a tube is set in the direction of the upper airway while ventilating the lungs. However, although the results of nasal administration of OVA to a mouse model for allergic rhinitis to OVA and measuring nasal resistance after 24 hours are disclosed, detailed methods and nasal hypersensitivity are not described.

  Furthermore, in Non-Patent Document 3, nasal cavity resistance (Rna) measurement by the IPG method of performing tracheostomy measurement is likely to be affected by anesthesia and surgical invasion, and there are problems in terms of experimental efficiency, It has been selected to evaluate changes in respiratory frequency using the non-invasive WPG method, and the IPG method has not been put to practical use.

  In allergic rhinitis, hypersensitivity of the nasal cavity is enhanced as in asthma, and it is necessary to measure nasal resistance in the upper respiratory tract, and development of a device capable of performing such measurement is urgently needed.

  The present invention has been made in consideration of the above conventional problems and demands from medical fields, and evaluates upper and lower airway hyperresponsiveness in a mouse model of allergic airway inflammation such as allergic rhinitis and asthma. A measuring method and a measuring apparatus using the measuring method are provided.

  The airway resistance measuring method according to the present invention, which has solved the above problems, is a method for measuring airway resistance using a mouse model of allergic airway inflammation such as allergic rhinitis and asthma, and the larynx and pharynx of the mouse model are decapitated. The method is characterized in that the posterior nostril cannula is inserted into the posterior nostril to measure nasal resistance in the upper airway.

  In addition, the method for measuring airway resistance according to the present invention, which has solved the above-mentioned problems, is characterized in that it opens the way to independently and concurrently measuring the nasal resistance in the upper airway and the pulmonary airway resistance in the lower airway. .

  The insertion of the posterior nostril cannula into the posterior nostril is preferably by inserting a tube into the posterior nostril and packing it to ensure a scavenging route into the nasal cavity and facilitate scavenging of the nasal cavity.

  It is preferable that the tube (intubation tube) inserted into and packed in the above-mentioned back nostril be indwelled so as to be able to scavenge toward the upper airway.

  It is preferable that the measurement to perform independently and concurrently the nasal resistance to the upper airway and the pulmonary airway resistance to the lower airway is generated by stimulating the upper airway and / or the lower airway.

  It is preferable that the measurement of the upper airway load and the sensitivity to the lower airway load be evaluated by the methacholine load, respectively.

  The airway resistance measuring device according to the present invention, which has solved the above-mentioned problems, is an airway resistance measuring device using allergic airway inflammation mouse model such as allergic rhinitis and asthma, and the constitution of the airway resistance measuring device is It consists of a nasal cavity resistance measurement part which measures the nasal cavity resistance by the load to the upper airway direction, and a lung resistance measurement part which measures the lung resistance by the load to the lower airway direction. In addition, since each route is composed of a nebulizer and a ventilator for loading, the nasal cavity resistance measuring unit and the pulmonary resistance measuring unit are independent, and the upper airway and lower airway resistance are independently established. It is characterized in that it can be correctly determined and measured in parallel.

  By using the method for measuring airway resistance of the present invention, it is possible to directly measure nasal resistance in the upper airway, so establishing a protocol for examining nasal hypersensitivity that could not be demonstrated so far. It becomes possible.

  In addition, since the airway resistance measuring method of the present invention can contribute to independently and concurrently measuring the nasal resistance to the upper airway and the lung airway resistance to the lower airway, an allergy based on upper airway hypersensitivity as an index. As well as contributing to the elucidation of the mechanism of allergic rhinitis, it also contributes to the elucidation of the relationship between the upper and lower airways.

  Furthermore, by using the airway resistance measuring device according to the present invention, nasal resistance in the upper airway can be measured and analyzed independently from pulmonary airway resistance in the lower airway, and this leads to the development of drug discovery for allergic rhinitis. Can be expected.

  The embodiments of the present invention necessary to explain the drawings are defined as follows.

  The embodiment of the present invention is grasped as a measurement method invention, and is defined as a method of measuring airway resistance using a mouse model of allergic airway inflammation such as allergic rhinitis and asthma. In the embodiment of the present invention, as the airway resistance measuring method, after the larynx and the pharynx of the mouse model are necropsied, the posterior nostril cannula is inserted into the posterior nostril to measure the nasal resistance in the upper airway. There is a mode in which another cannula is inserted also in the lower airway direction, and the nasal resistance in the upper airway and the lung airway resistance in the lower airway are measured independently and in parallel.

  In addition, an embodiment of the present invention includes an airway resistance measurement device used when performing the above airway resistance measurement method. The airway resistance measuring apparatus comprises a nasal cavity resistance measuring unit for measuring a load in the upper airway direction and a nasal cavity resistance, and a pulmonary airway resistance measuring unit for measuring a load in the lower airway direction and a pulmonary airway resistance, each with a nebulizer And a ventilation device, wherein the nasal cavity resistance measurement unit and the lung airway resistance measurement unit can be independently and parallelly measured.

FIG. 3 is an overall conceptual view showing measurement of nasal cavity resistance in the upper airway and pulmonary airway resistance in the lower airway in the airway resistance measuring method according to the embodiment of the present invention. It is a drawing substitute photograph which shows the airway resistance measuring device concerning the embodiment of the present invention. The upper right picture shows the mouse attached to the airway resistance measuring device. Longitudinal cross-sectional view (a) of the posterior nostril cannula of the cannula used in the method of measuring airway resistance according to an embodiment of the present invention, transverse cross-sectional view (b) of the posterior nostril cannula, posterior nostril cannula and cannula inserted in the lower airway direction The drawing substitute photograph (c) is shown. The airway resistance measurement method which concerns on embodiment of this invention WHEREIN: The schematic diagram of the position of tube indwelling and insertion method to a nostril is shown. (A) and (b) show a sagittal view of the mouse head and a view in which a larynx is incised and a nostril is necropsied, respectively. In the airway resistance measuring method according to an embodiment of the present invention, a drawing substitute photograph (A) actually showing the posterior nostril of the mouse after necropsy, a drawing substitute photograph (B) inserting the posterior nostril cannula into the rear nostril, and upper airway direction The drawing substitute photograph (c) which inserted each different cannula in the lower airway direction is shown. In accordance with the airway resistance measuring method according to the embodiment of the present invention, an example of measurement results when the nasal cavity resistance of the upper airway and the lung airway resistance of the lower airway are independently measured by applying methacholine loading in the direction of the nasal cavity is shown. .

  Hereinafter, an airway resistance measuring method and an airway resistance measuring apparatus according to an embodiment of the present invention will be described with reference to the drawings. In the following description, the same components are denoted by the same reference numerals.

  The airway resistance measuring method and airway resistance measuring apparatus of the present invention are the measuring method and the measuring apparatus for allergic airway inflammation mouse models such as allergic rhinitis and asthma. A disease mouse model is a mouse used to develop researches leading to elucidation of the disease state of the disease and methods for preventing or treating the disease, and mice used in the present invention cause allergic airway inflammation. . A BALB / c mouse (female, 6-12 weeks old) is used as a mouse model that develops allergic rhinitis, asthma and the like, and the protocol will be described below.

(Preparation of mouse model)
Allergic rhinitis and asthmatic mice were prepared according to the following procedures.
(I) Sensitization of OVA: Normal BALB / c mice were administered intraperitoneally with OVA (50 μg / animal) and aluminum hydroxide (2 mg / animal) to perform systemic sensitization to OVA (days 0 and 14) Day).
(Ii) OVA challenge to the airway: OVA challenge to the airway can produce an allergic rhinitis model with asthma. With regard to the administration period and timing, there is no fixed method since it is necessary to consider the experimental design and purpose. As a specific example, nasal administration of 0.5% OVA (left and right nasal cavity: 10 μg × 2) is performed. The administration period is: continuous administration of 0.5% OVA continuously for 8 days (from day 21 to day 28) in the acute model, or once weekly administration of the 0.5% OVA in the chronic model Is administered for 5 weeks or more (5 or more times on days 21, 28, 35, 42, 49).

(Airway resistance measurement)
The airway resistance measurement using a mouse model is performed using Buxco airway analysis system MaxII (manufactured by DSI). The specific procedure is shown below.
(A) Anesthesia in mice: Anesthesia with a cocktail of 10% medetomidine and 10% ketamine (5 mL / kg body weight), and surgical treatment after sedation immobilization.
(B) Intubation of lower respiratory tract: First, cut the skin of the front neck of the mouse, and fully dissect the larynx and trachea. To ensure ventilation, a 20-gauge cannula is tracheally intubated in the lower airway direction and secured with sutures to prevent it from falling off.
(C) Intubation into the upper airway: As shown in FIGS. 4 and 5, a facet is added to the anterior wall of the larynx, a part of the hyoid bone is removed, and the pharyngeal cavity is placed under the clear vision. A portion is removed and the posterior nostril is completely decapitated. After the posterior nostril is confirmed, the posterior nostril cannula is inserted while rotating (the outer peripheral surface is processed into a screw shape) and the posterior nostril is completely packed. Also secure the anterior neck and the cannula with a suture so that the tube will not come off. The surgical procedure is performed under a stereomicroscope because a fine surgical procedure is required.
(D) Fixation to airway resistance device: Place the mouse on the measurement stand, connect the cannula inserted in the upper airway direction and the lower airway direction with the measurement device, and start ventilation. A muscle relaxant (5 mL / kg; 1% pancuronium bromide) is intraperitoneally administered in order to avoid the influence of nonspecific movement (body movement), and it is observed until it stabilizes (about 5 minutes). Since the success or failure of the packing of the back nostril greatly affects the experimental results, perform a trial nebulizer in the upper airway direction, and confirm that the smoke-like mist escapes from the nasal cavity direction to the nostril And check that there is no leak), start the measurement.
(E) Setting of ventilation conditions: Ventilation conditions in the lower airway direction are performed under general conditions (specific conditions are 150 ventilations / min, volume 10 μL / g, PEEP ₂ cm H ₂ O). On the other hand, the upper airway direction is measured under the condition of ventilation number 100 times / min, volume 2.5-5 μL / g, without PEEP. For ventilation toward the lower airway, insert the posterior nostril cannula into the posterior nostril to measure nasal resistance in the upper airway, or independently and concurrently measure nasal resistance in the upper airway and pulmonary airway resistance in the lower airway It is also necessary if you Unlike the lower airway (lung), it is not necessary to ventilate the upper airway, so mere exhausting is sufficient.
(F) Measurement of airway hyperresponsiveness: With regard to the determination of airway hyperresponsiveness, methacholine is loaded for examination. For measurement of nasal hypersensitivity and impact on the lower respiratory tract, use a nebulizer in the nasal direction and use 10 μL of each concentration of methacholine (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 mg The examination is carried out by measuring the nasal resistance and the lung airway resistance by measuring each for 2 minutes. On the other hand, also for lung hypersensitivity and upper respiratory tract effects, similarly, methacholine (0, 1.5, 3.125, 6.25, 12.5, 25, 50 mg / mL) at each concentration in the lung direction Load and study. Although hypersensitivity has been examined using methacholine, various applications can be selected by changing the reagent.
(G) Analysis method: The airway resistance value at each concentration is evaluated using FinePoints analysis software (manufactured by Buxco). After loading, measurements are taken for 2 minutes at 2 second intervals, and the average value is calculated as airway resistance. If airway resistance rises at low concentrations compared to normal mice, it is determined that there is airway hyperresponsiveness. Thus, in the diseased mouse model, airway resistance increases at lower concentrations compared to normal mice.

(Embodiment)
FIG. 1 is an overall conceptual view showing a method of measuring airway resistance according to an embodiment of the present invention. The airway resistance measurement is performed as described in (A) to (G) above, after the larynx and pharynx of the mouse 1 are necropsied, the posterior nostril cannula 2 is inserted into the posterior nostril in the nasal direction, and the lung in the lower airway direction The lower airway cannula 3 is inserted into the airway to measure airway resistance. The airway resistance measuring device 11, the nebulizer 14, the ventilator 15, and the sensor 16 shown in FIG. 1 will be described based on FIG.

  FIG. 2 is a drawing-substituting photograph showing an airway resistance measuring device according to an embodiment of the present invention. The upper right shows the mouse 1 attached to the device. The airway resistance measuring device 11 includes a nasal cavity resistance measuring unit 12 that measures nasal cavity resistance due to stimulation in the upper airway direction, a lung airway resistance measuring unit 13 that measures pulmonary airway resistance due to stimulation in the lower airway direction, and a nebulizer 14 , And a ventilator 15.

  The nasal cavity resistance measurement unit 12 is connected to the rear nostril cannula 2 so that the upper airway can be loaded with methacholine to measure the nasal cavity resistance via the sensor 16. On the other hand, the lung airway resistance measurement unit 13 is connected to the lower airway cannula 3 indwelling in the lower airway direction, and the lower airway is loaded with methacholine to measure the lung airway resistance via the sensor 16. It is possible to In addition, methacholine is a reagent generally used to examine the hypersensitivity of the lung, and the sensitivity of contraction of the bronchus can be objectively evaluated. For example, people with asthma respond to much less methacholine than healthy people.

  The nebulizer 14 sprays methacholine, which is a stimulant, to the airway of the mouse 1, and is not particularly limited in terms of performance and size, and a commercially available sprayer can be used. The nebulizer 14 may be installed at one place in each of the upper airway direction and the lower airway direction with respect to the airway resistance measuring device 11. However, the nebulizer 14 may be installed in both the upper airway direction and the lower airway direction. It is also possible to install.

  The ventilator 15 promotes the exhaust of the upper airway and the lower airway with respect to the spray supply of methacholine to the airway of the mouse 1, and is not particularly limited in terms of performance and size, and a commercially available ventilator may be used. It can be used.

  The airway resistance measurement can be performed by locally applying methacholine to the upper airway to measure changes in nasal resistance associated with nasal discharge from the nasal gland of mouse 1 and mucosal swelling. It is also possible to perform parallel measurements of upper airway (nasal) resistance and lower airway (lung airway) resistance due to methacholine loading towards the lower airway. That is, by applying methacholine to the upper airway, it is possible to measure a change in nasal resistance associated with an increase in nasal discharge from the nasal glands of mouse 1 or a pulmonary airway resistance associated with smooth muscle contraction in the lower airway of mouse 1 The change of can also be measured.

  In addition, the airway resistance measuring device 11 of the present invention can also individually examine the influence on the lower airway resistance by applying the upper airway load and the influence on the upper airway resistance by applying the lower airway load. it can. With these measurements, it is also possible to investigate the effect of lower airway load on lower airway resistance, which normally would not be considered, or the effect of lower airway load on upper airway resistance. In addition, the airway resistance measuring device 11 of the present invention can simultaneously measure the upper airway resistance and the lower airway resistance by simultaneously loading the upper airway direction and the lower airway direction. By this measurement, it is possible to immediately evaluate the inflammatory condition occurring in the upper and lower airways.

  FIG. 3 shows the cannula used. (A) is a longitudinal sectional view of the back nostril cannula 2, (b) is a transverse sectional view of the back nostril cannula 2, and (c) is a drawing of the lower airway cannula 3 inserted into the posterior nostril cannula 2 and the lung airway in the lower airway direction. Show a substitute photo. The material and shape of the cannula are not particularly limited as long as they can be easily inserted into the posterior nostril and maintain sufficient strength, but the workability of insertion of the posterior nostril cannula 2 into the posterior nostril is improved From the point of view, it is preferable to use the posterior nostril cannula 2 shaped as shown in (a). Specifically, it is preferable to use a commonly used 20 G metal cannula for tracheal intubation attached with a silicone Papara II type tube used for tympanic dissection treatment of childhood effusion otitis media. The material of the packing is not limited as long as it has elasticity. In order to stably perform the insertion operation into the nostril, it is desirable to form a screw-like spiral groove. The cannula is generally a pipe-like medical device used for injection of a drug solution, discharge of body fluid, and passage of air at tracheostomy, etc. by inserting it into a body cavity, a blood vessel or the like.

  FIG. 4 is a surgical schematic for placing the posterior airway cannula 2 in the posterior nostril and the lower airway cannula 3 in the lower airway in the method of measuring airway resistance according to an embodiment of the present invention. (A) shows a sagittal cross-section of the mouse head, (b) shows the front after the post-nostril necropsy. A facet is added to the anterior laryngeal wall, a portion of the hyoid is resected, the pharyngeal cavity is under sight, a portion of the vocal cords or posterior laryngeal wall is resected, and the posterior nares are completely necrotized.

  FIG. 5 shows an actual situation in which the posterior nostril cannula is placed in the posterior nostril. (A) is a drawing-substituting photograph of actually decapsulating the back nostril of a mouse, (b) is a drawing-substituting photograph of inserting the back nostril cannula 2 in the back nostril, (c) is an upper airway direction and a lower airway direction Fig. 13 is a photograph as a substitute of a drawing in which separate cannulas 2 and 3 are inserted respectively. A specific surgical procedure is to perform tracheal intubation (cannula 3) in the lower airway to ensure ventilation in the lower airway (in the figure, the procedure is reversed for clarity). For confirmation of the posterior nostril, a longitudinal section is added to the front of the larynx of the mouse, partial tissue of the larynx and pharynx is excised, and the posterior nostril is necropsied. By placing the back nostril under a clear view, tube insertion (insertion of the back nostril cannula 2) can be easily performed. By inserting the posterior nostril cannula 2 into the posterior nostril after necropsy, air tightness between the inner peripheral surface of the posterior nostril and the outer peripheral surface of the posterior nostril cannula 2 can be ensured, and the airtight state of the posterior nostril and scavenging It works effectively in that it is possible to secure a space (for avoiding collapse), and stable data can be obtained.

  6 (a) and 6 (b) show the measurement results of upper airway (nasal cavity) resistance and lower airway (lung airway) resistance by methacholine loading in the upper airway direction. Methacolin was loaded in the nasal direction, and simultaneous measurements of nasal resistance (a) and pulmonary airway resistance (b) were performed. A comparison study is conducted between three groups in which a normal mouse model (negative control: NC), an airway inflammation model (positive control: PC) and a PC mouse are pretreated with Compound X (a drug under development). In (c), the change of the nasal cavity histology before and after loading with methacholine is shown. After loading, the nasal cavity volume decreases due to swelling of mucous membrane and nasal discharge.

  It was found that methacholine loading in the nasal cavity not only increases nasal resistance but also increases pulmonary airway resistance in airway allergic inflammation mice (positive control: PC). In addition, pretreatment of the asthmatic mice with compound X suppressed the increase in nasal resistance and pulmonary airway resistance. That is, not only nasal hypersensitivity was demonstrated, but the existence of the interaction between the upper and lower airways was confirmed. Based on these comprehensive findings, the usefulness of the method for measuring airway resistance of the present invention in drug discovery research was not only confirmed that pretreatment with compound X showed a reducing effect on nasal hypersensitivity, but also upper airway It was found that the effects on the lower respiratory tract were reduced. In (c), it can be inferred that the nasal cavity volume of the mouse decreased and the nasal cavity resistance value increased due to the methacholine loading compared with before the methacholine loading. Thus, these results demonstrate nasal hypersensitivity due to methacholine loading.

  The airway resistance measurement method according to the embodiment of the present invention can choose to load both the upper and lower airways, or to load either the upper or lower airways. By this measurement method, the inflammatory condition occurring in the upper and lower airways can be evaluated simultaneously or separately.

  In addition, it is preferable to locally apply methacholine to the upper and lower airways in order to measure hypersensitivity in the upper and lower airways.

Reference Signs List 1 mouse 2 back nostril cannula 3 lower airway cannula 11 airway resistance measuring device 12 nasal cavity resistance measuring unit 13 lung airway resistance measuring unit 14 nebulizer 15 ventilation device 16 sensor

Claims (6)

A airway resistance measuring method using the allergic airway inflammation mouse model of allergic rhinitis and asthma,
After necropsy of the larynx and pharynx of the mouse model, a posterior nostril cannula is inserted into the posterior nostril to measure nasal resistance in the upper respiratory tract ,
Insertion of nasal cannula after the said rear nostrils, packing by inserting a screw-shaped tube rear nostril, and airway resistance measuring method comprising der Rukoto shall ensure a space for exhaust . A airway resistance measuring method using the allergic airway inflammation mouse model of allergic rhinitis and asthma,
After necropsy of the larynx and pharynx of the mouse model, a posterior nostril cannula is inserted in the posterior nostril, and a cannula is inserted in the direction of the lower airway to independently control the nasal resistance in the upper airway and the pulmonary airway resistance in the lower airway. Measure in parallel ,
A method of measuring airway resistance comprising inserting a screw-like tube into the posterior nostril and packing the posterior nostril cannula into the posterior nostril, and securing a space for exhaustion . The airway resistance measuring method according to claim 1 or 2 , wherein the tube is placed in the direction of the upper airway.   The airway according to claim 2, wherein the measurement in which the nasal resistance in the upper airway and the pulmonary airway resistance in the lower airway are independently and parallelly produced is by stimulating the upper airway and / or the lower airway. How to measure resistance. 5. The airway resistance measurement method according to claim 4 , wherein the stimulation to the upper airway and the lower airway is due to a methacholine loading, respectively. A airway resistance measuring apparatus using the allergic airway inflammation mouse model of allergic rhinitis and asthma,
The airway resistance measuring device includes a nasal cavity resistance measuring unit that measures stimulation in the direction of the upper airway and nasal cavity resistance;
A lung airway resistance measurement unit that measures stimulation in the lower airway direction and lung airway resistance;
A nebulizer, a ventilating device , and a posterior nostril cannula which is a screw-like tube which is inserted into the posterior nostril for packing and space for evacuation ;
An airway resistance measuring device characterized in that the nasal cavity resistance measuring unit and the lung airway resistance measuring unit can be independently and concurrently measured.