Non-exposed airway instillation administration method for mouse lung specific administration

Abstract

The invention discloses a non-exposed airway instillation administration method for mouse lung specific administration, which comprises the following steps of: 1, preparing different types of drugs to be administered, namely a hydrophilic drug, a hydrophobic drug and a cellular drug; 2, the mouse is placed in a matched anesthesia device, and anesthesia treatment is conducted on the mouse; then the anesthetized mouse is placed on a matched hanging device, and the body of the mouse naturally droops; 3, the tongue of the mouse is pulled out through bent tweezers until the mouse feels resistance, and the action can open the epiglottis and expose the airway; 4, applying a medicine to the root of the tongue by using a pipettor, and covering the nasal cavity of the mouse by using the finger wearing the glove; meanwhile, the tongue of the mouse is maintained to extend outwards and the nasal cavity is covered for 10 breaths, and administration is completed; compared with the common lung tissue drug delivery technology at present, the non-exposed airway instillation drug delivery method provided by the invention has remarkable comprehensive advantages, and low-cost, non-invasive and multi-time mouse lung tissue specific drug delivery of cell / non-cell drugs can be realized.

Description

Technical Field

[0001] The invention relates to the technical field of lung tissue drug delivery, in particular to a non-exposed airway instillation drug delivery method for mouse lung-specific drug delivery. Background Art

[0002] Infectious or non-infectious lung diseases such as pneumonia, lung cancer, pulmonary fibrosis, acute respiratory distress syndrome, and chronic obstructive pulmonary disease have become major public health issues that threaten human health. The first step in developing new and highly effective therapeutic drugs for lung diseases is to establish corresponding mouse models and administer drugs to them for preclinical experiments. However, both the establishment of lung disease models and the administration of new therapeutic drugs rely on appropriate lung tissue administration techniques.

[0003] Currently, commonly used lung tissue drug delivery technologies include intranasal delivery, exposed airway delivery, and aerosol delivery.

[0004] Intranasal administration is a method of delivering drugs to the respiratory tract and lungs through the nasal cavity. It mainly utilizes the rich blood vessels and lymphatic vessels in the nasal mucosa. After the drug is absorbed by the nasal mucosa, it can enter the lungs along the blood circulation under the mucosa to exert its effect.

[0005] Exposure airway drug delivery is usually used in specific experimental research scenarios (such as animal experiments to study the mechanism of lung diseases, drug effects, etc.) or in some special clinical treatment situations. It requires surgery and other means to open and expose airway parts such as trachea and bronchi, and then directly and accurately inject the drug into the airway so that the drug can directly act on the lung tissue.

[0006] Nebulization drug delivery is a drug delivery method that uses a nebulizer to transform a drug solution into tiny mist particles to form an aerosol state. The user inhales the drug-containing aerosol into the respiratory tract and even the lungs through breathing. The mist particles produced by atomization are of appropriate size and can penetrate into the bronchi at all levels and even the alveoli with the inhalation airflow, so that the drug can be widely and relatively evenly distributed in the lung tissue, achieving the effect of local treatment of lung diseases (such as bronchitis, pneumonia, etc.).

[0007] Although the above-mentioned administration methods can achieve the purpose of drug administration and treatment, they have some defects. For example, intranasal administration cannot measure the drug that actually enters the lung tissue due to the loss of drugs in the nasal cavity and sinuses; exposed airway administration requires surgery on mice and damages the airway, and secondary administration can cause the death of mice; in addition, atomization administration relies on expensive atomization devices and cannot deliver cell drugs to lung tissue (atomization shear force can cause cell apoptosis). Relatively speaking, the non-exposed airway instillation administration method described in the present invention has significant advantages, and the required devices are all essential infrastructure for biomedical laboratories, which can achieve low-cost, non-invasive, and multiple mouse lung tissue-specific administration of cell / non-cell drugs. Summary of the invention

[0008] The purpose of the present invention is to provide a non-exposed airway instillation method for mouse lung-specific drug delivery, aiming to improve the problem that the lung tissue drug delivery technology needs to be improved.

[0009] The present invention is achieved by: a non-exposed airway instillation method for mouse lung-specific administration, comprising:

[0010] Step 1: Prepare different types of drugs to be administered, namely hydrophilic drugs, hydrophobic drugs, and cell-based drugs;

[0011] Step 2: placing the mouse in a matching anesthesia device and anesthetizing the mouse; then placing the anesthetized mouse on a matching hanging device to allow its body to droop naturally;

[0012] Step 3: Use curved forceps to pull out the mouse's tongue until you feel resistance. This action will open the epiglottis and expose the airway.

[0013] Step 4: Use a pipette to apply the drug to the root of the tongue and cover the mouse's nasal cavity with a gloved finger; at the same time, keep the mouse's tongue extended and the nasal cavity covered for 10 breaths to complete the drug administration.

[0014] Preferably, the hydrophilic drug is dissolved in sterile saline and then filtered through a 0.22 μm filter membrane for sterilization before use; the hydrophobic drug is dispersed in sterile saline containing 30% corn oil, and ultrasonically dissolved to form a suspension before use; the cell-based drug is resuspended in a serum-free culture medium in a clean bench and the concentration should be less than 10 5 / μL; the single administration volume of hydrophilic drugs, hydrophobic drugs, and cellular drugs should be less than 50μL, and the interval between multiple administrations should be greater than 6h.

[0015] A matching anesthesia device comprises a base, wherein the base comprises a bottom tube and a supporting net, wherein the top of the bottom tube is arranged to be open, and a side tube is arranged on the side in communication, and the supporting net is detachably arranged at the opening of the bottom tube; a transparent tube body is detachably and sealedly arranged above the base, and a top cover is sealedly arranged on the top of the transparent tube body.

[0016] Preferably, a rubber stopper is provided at the top; an external thread groove is provided on the upper side surface of the bottom cylinder, a threaded structure is provided at the bottom of the transparent tube body, and the bottom is inserted into the external thread groove; an internal thread groove is further provided on the upper side surface of the bottom cylinder, the internal thread groove is located inside the external thread groove, an annular plate is provided on the outer side of the support net, and the annular plate is threadedly installed at the internal thread groove; two circles of sealing rings are provided inside and outside the lower side surface of the top cover, and the two circles of sealing rings are distributed on both sides inside and outside the transparent tube body.

[0017] A supporting suspension device includes a base, a bracket is detachably provided on the base, two groups of limiting plate groups are symmetrically provided in the middle of the bracket, and a suspension mechanism is provided at the top; the suspension mechanism includes a suspension rope, both ends of the suspension rope are installed at the top of the bracket, and the middle part can be suspended at the upper front teeth of the mouse; one end of the two groups of limiting plate groups adjacent to each other contacts the body of the mouse.

[0018] Preferably, the bracket includes a bottom ring plate, two vertical plates and two U-shaped plates. The bottom ring plate is threadedly installed on the bottom cylinder of the base. The two vertical plates are symmetrically and fixedly provided above the bottom ring plate. The two U-shaped plates are respectively fixedly provided at the tops of the two vertical plates; an installation groove is provided at the bottom of the bottom cylinder, a pull plate and a support plate are provided at the installation groove, the pull plate is installed at the end of the support plate, and can extend out of the bottom cylinder.

[0019] Preferably, the suspension mechanism further includes a first end plate and a second end plate. Both the first end plate and the second end plate are set as I-shaped structures and are adapted to the U-shaped plates and are installed on the U-shaped plates at the same time; a tapered through hole is provided on the first end plate, a frustum hole is provided on the second end plate, the suspension rope passes through the frustum hole, and the end extends into the through hole. At the same time, the end ball installed at the end of the suspension rope is located at the end with a larger size of the through hole.

[0020] Preferably, a support frame is fixedly provided on the side of the second end plate away from the first end plate. The support frame is set as a U-shaped structure, and an adjustment screw is threadedly penetrated through the middle. A frustum column is provided at the end of the adjustment screw, and the frustum column can extend into the frustum hole to squeeze the suspension rope; a convex column is provided at the end of the support frame protruding from the second end plate, and a groove is provided on the side wall of the U-shaped plate, and the convex column is arranged in the groove.

[0021] Preferably, the limiting plate group includes a U-shaped plate, a first clamping plate, a second clamping plate and an adjustable plate. The first clamping plate and the second clamping plate are connected by bolts and are distributed on both sides of the vertical plate, and the bolts penetrate through the sliding holes of the vertical plate. The adjustable plate is located on the side where the two vertical plates are close to each other. The vertical plate penetrates through the U-shaped plate, and a pressing arc plate is fixedly provided at the end. A first magnet and a second magnet that repel each other are respectively provided on the adjacent sides of the pressing arc plate and the adjustable plate.

[0022] Preferably, the first plate body and the second plate body are staggered and arranged on one side where the first splint and the second splint are close to each other, and the first plate body and the second plate body are both arranged in the sliding hole; baffles are fixedly arranged on the upper and lower vertical edges of the first splint, and the plate passes through the gap formed by the two baffles; a limiting rod is fixedly arranged at one end of the adjustable plate, and an adjusting screw is passed through the other end through a bearing connection, the adjusting screw thread passes through the first splint, and the limiting rod passes through the first splint.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The non-exposed airway instillation drug delivery method provided by the present invention has significant comprehensive advantages over the currently commonly used lung tissue drug delivery technology, and can achieve low-cost, non-invasive, and multiple mouse lung tissue-specific delivery of cellular / non-cellular drugs.

[0025] 2. The matching anesthesia device provided by the present invention can extract excess anesthetic drugs in the anesthesia device after the mouse is anesthetized and before the mouse is taken out, so as to avoid leakage of anesthetic drugs and cause discomfort to the experimenter, so as to facilitate the experimenter to conduct experiments multiple times.

[0026] 3. The matching suspension device provided by the present invention can lift the mouse to a vertical state after the suspension mechanism hooks the mouse's upper incisors, and then control the mouse's body with the cooperation of two groups of restriction plates so that the mouse is placed stably, providing convenience for completing non-exposed airway instillation drug administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the structure of the anesthesia device of the present invention;

[0028] Figure 2 is a first structural schematic diagram of the base of the present invention;

[0029] Figure 3 It is a structural schematic diagram of the bottom tube of the present invention;

[0030] Figure 4 It is a structural schematic diagram of the support net of the present invention;

[0031] Figure 5 It is a schematic structural diagram of the transparent tube body of the present invention;

[0032] Figure 6 It is a schematic diagram of the structure of the top cover of the present invention;

[0033] Figure 7 It is a structural schematic diagram of the suspension device of the present invention;

[0034] Figure 8 is a second structural schematic diagram of the base of the present invention;

[0035] Fig. 9 It is a schematic diagram of the structure of the support of the present invention;

[0036] Fig.10 It is a structural schematic diagram of the suspension mechanism of the present invention;

[0037] Fig.11 It is a schematic diagram of the structure of the first end plate and the suspension rope of the present invention;

[0038] Fig.12 is a schematic structural diagram of a second end plate of the present invention;

[0039] Fig.13 It is a structural schematic diagram of the restriction plate assembly of the present invention;

[0040] Fig.14 It is a schematic diagram of the structure of the first clamping plate, the second clamping plate and the adjustable plate of the present invention;

[0041] Fig.15 The aim was to compare the drug distribution in lung lobes after intranasal administration, exposed airway administration, nebulization administration, and non-exposed airway instillation administration;

[0042] Fig.16 It is to compare the drug delivery efficiency of intranasal administration, exposed airway administration, nebulization administration and non-exposed airway instillation administration for non-cellular drugs;

[0043] Fig.17 It is to compare the drug delivery efficiency of intranasal administration, exposed airway administration, nebulization administration and non-exposed airway instillation administration of cell-based drugs;

[0044] Fig.18 It is to compare the survival of mice after repeated administration of intranasal administration, exposed airway administration, nebulization administration, and non-exposed airway instillation administration;

[0045] Fig.19 is a schematic diagram of the mouse anesthesia facility;

[0046] Fig. 20 This is a schematic diagram of the mouse anesthesia operation;

[0047] Fig.21 is a schematic diagram of the mouse hanging operation;

[0048] In the figure: 1, base; 11, bottom tube; 111, side tube; 112, rubber plug; 113, external thread groove; 114, internal thread groove; 12, support net; 121, annular plate; 13, mounting groove; 14, pull-out plate; 15, support plate; 2, transparent tube body; 21, thread structure; 3, top cover; 31, sealing ring; 4, bracket; 41, bottom ring plate; 42, vertical plate; 43, sliding hole; 44, U-shaped plate; 45, groove; 5, limiting plate group; 51, plate; 511, pressure arc plate ; 512, first magnet; 52, first clamping plate; 521, first plate body; 522, baffle; 53, second clamping plate; 531, second plate body; 54, adjustable plate; 541, second magnet; 542, adjusting screw; 543, limiting rod; 6, suspension mechanism; 61, suspension rope; 611, end ball; 62, first end plate; 621, through hole; 63, second end plate; 631, threaded rod; 632, frustum hole; 633, boss; 634, support frame; 635, frustum column. DETAILED DESCRIPTION

[0049] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0050] The following is a further description with reference to the accompanying drawings and specific embodiments:

[0051] Example 1

[0052] like Figure 15-21 As shown, a non-exposed airway instillation method for lung-specific drug delivery in mice comprises the following steps

[0053] Operation 1: Prepare the drugs according to their type: Dissolve the hydrophilic drugs in sterile saline and filter them through a 0.22 μm filter membrane for sterilization; Disperse the hydrophobic drugs in sterile saline containing 30% corn oil and dissolve them with ultrasound to form a suspension; Resuspend the cell-based drugs in serum-free culture medium in a clean bench and set aside. The concentration should be less than 10 5 Regardless of the type of drug, the single dose volume should be less than 50 μL, and the interval between multiple doses should be greater than 6 hours.

[0054] Procedure 2: In a fume hood, add 2 mL of isoflurane onto a cotton ball, place it on the bottom of a 1L beaker and cover the beaker with a glass plate. Alternatively, place the mouse in a matching anesthesia device and anesthetize the mouse. The initial breathing pattern of the mouse is rapid and shallow. After about 30 seconds, the mouse's breathing rate is close to 1 breath / 2 seconds, indicating that the mouse has been fully anesthetized.

[0055] Operation 3: Equip a pair of curved tweezers and a 100μL pipette with a pipette tip to absorb the drug to be administered as described in Operation 1 and place it on the side of the hanging device.

[0056] Operation 4: Take out the fully anesthetized mouse and hang its upper incisors on the hanging device, making sure that the mouse's body hangs naturally and its lower limbs and tail do not touch the experimental table.

[0057] Operation 5: Use curved forceps to gently pull out the mouse tongue until you feel slight resistance. This action opens the epiglottis and exposes the airway. At the same time, use a pipette to apply the drug to the root of the tongue and immediately cover the mouse's nasal cavity with a gloved finger. Maintain the mouse's tongue out and cover the nasal cavity for 10 breaths to complete the drug administration.

[0058] Operation VI: Remove the mouse and place it back in its cage to monitor its recovery from anesthesia.

[0059] In order to fully compare the difference between this administration method and the existing administration method, multiple groups of experiments are set up below, and the results are averaged.

[0060] Experiment 1 compared the drug lung lobe distribution after intranasal administration, exposed airway administration, nebulization administration and non-exposed airway instillation administration.

[0061] 1. Experimental Materials

[0062] 1. Experimental Animals

[0063] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0064] 2. Experimental Equipment

[0065] Electronic balance, constant temperature water bath, small animal anesthesia machine, small animal nebulizer

[0066] 3. Experimental Reagents

[0067] Isoflurane, Evans blue, normal saline

[0068] 2. Experimental Methods

[0069] 1. Prepare 2% Evans blue-physiological saline solution as an indicator;

[0070] 2. 20 μL of 2% Evans blue-normal saline solution was delivered to the lungs of C57BL / 6 mice by intranasal administration (after isoflurane anesthesia, an equal amount of drug solution was dripped into the left and right nasal cavities of mice at a uniform speed), exposed airway administration (after isoflurane anesthesia, the neck skin was cut to expose the airway, the drug solution was injected after airway intubation, and the wound was then sutured), nebulization administration (the mice were placed in an induction box, the drug solution was added to the nebulizer cup, the power was turned on, and the corresponding administration time and concentration were set), and non-exposed airway instillation administration;

[0071] 3. The mice were killed after 30 minutes, and their left lung lobes were taken for photographic analysis.

[0072] 3. Experimental Results

[0073] Evans blue is a commonly used azo dye preparation, and the dark blue mark caused by it can be used to reflect the distribution of drugs in the lung lobes of mice after administration in different ways. Fig.15 As shown, the dark blue marks in the non-exposed airway instillation, nebulization and exposed airway administration groups are more numerous and more evenly distributed, indicating that non-exposed airway instillation, nebulization and exposed airway administration can all achieve uniform distribution of the drug in the lung lobes, which is better than intranasal administration.

[0074] Experiment 2 compared the drug delivery efficiency of intranasal administration, exposed airway administration, nebulization administration, and non-exposed airway instillation for non-cellular drugs.

[0075] 1. Experimental Materials

[0076] 1. Experimental Animals

[0077] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0078] 2. Experimental Equipment

[0079] Electronic balance, constant temperature water bath, small animal anesthesia machine, small animal nebulizer, paraffin embedding machine, paraffin baking machine

[0080] 3. Experimental Reagents

[0081] Isoflurane, bleomycin, saline, xylene, anhydrous ethanol, 10% neutral formalin, Masson staining kit

[0082] 2. Experimental Methods

[0083] 1. Prepare 3.5 mg / mL bleomycin-saline solution as a non-cellular model drug;

[0084] 2. 20 μL of 3.5 mg / mL bleomycin-saline solution was delivered to the lungs of C57BL / 6 mice by intranasal administration (after isoflurane anesthesia, an equal amount of drug solution was dripped into the left and right nasal cavities of the mice at a uniform speed), exposed airway administration (after isoflurane anesthesia, the neck skin was cut to expose the airway, and the drug solution was injected after airway intubation), nebulization administration (the mice were placed in an induction box, the drug solution was added to the nebulizer cup, the power was turned on, and the corresponding administration time and concentration were set), and non-exposed airway instillation administration;

[0085] 3. The mice were killed after 3 weeks, and paraffin sections of their lung tissues were taken for Masson staining.

[0086] 3. Experimental Results

[0087] Bleomycin is a multi-component compound antibiotic of alkaline glycopeptide produced by Streptomyces verticillium. Administration of a certain concentration of bleomycin to lung tissue can induce DNA breakage in alveolar epithelial cells, cause cell apoptosis or necrosis, and induce excessive deposition of extracellular matrix and pulmonary fibrosis. Masson staining of paraffin sections of lung tissue of each group of mice can be used to evaluate the excessive deposition of extracellular matrix, which reflects the efficiency of bleomycin administration by different administration methods. Fig.16 As shown, the Masson staining of the non-exposed airway instillation group was darker than that of the nebulization and exposed airway administration groups, indicating that when non-cellular drugs are administered, the administration efficiency of non-exposed airway instillation is equivalent to that of nebulization and exposed airway administration, and is better than that of intranasal administration.

[0088] Experiment 3 compared the drug delivery efficiency of intranasal administration, exposed airway administration, nebulization administration, and non-exposed airway instillation administration of cell-based drugs.

[0089] 1. Experimental Materials

[0090] 1. Experimental Animals

[0091] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0092] 2. Experimental Equipment

[0093] Electronic balance, constant temperature water bath, small animal anesthesia machine, small animal nebulizer, cell culture box

[0094] 3. Experimental Reagents

[0095] Isoflurane, PBS, fetal bovine serum, DMEM medium

[0096] 2. Experimental Methods

[0097] 1. Take the logarithmic growth phase mouse lung cancer cell line LLC, digest it with trypsin, wash it with PBS, count it, and prepare it into 105 / μLL LCC cell-serum-free DMEM medium solution as a cell-based model drug;

[0098] 2. 20 μL of 105 / μL LLC cell-serum-free DMEM medium solution was delivered to the lungs of C57BL / 6 mice by intranasal administration (after isoflurane anesthesia, an equal amount of drug solution was dripped into the left and right nasal cavities of mice at a uniform speed), exposed airway administration (after isoflurane anesthesia, the neck skin was cut to expose the airway, and the drug solution was injected after airway intubation), nebulization administration (the mice were placed in an induction box, the drug solution was added to the nebulizer cup, and the power was turned on, and the corresponding administration time and concentration were set) and non-exposed airway instillation administration;

[0099] 3.4 weeks later, the mice were killed and their lung tissues were taken for photographic analysis.

[0100] 3. Experimental Results

[0101] The mouse lung cancer cell line LLC can be used to construct an orthotopic lung cancer model in mice. The number and volume of lung tumors in each group of mice reflect the efficiency of LLC cell administration in different administration methods. Fig.17 As shown, the number of lung tissue tumors in the non-exposed airway instillation group and the exposed airway administration group were greater and the total volume was larger, indicating that when administering cell-based drugs, the administration efficiency of non-exposed airway instillation and exposed airway administration is equivalent, and is better than intranasal administration and nebulization administration.

[0102] Experiment 4 compared the survival of mice after repeated administration of intranasal administration, exposed airway administration, nebulization administration, and non-exposed airway instillation administration.

[0103] 1. Experimental Materials

[0104] 1. Experimental Animals

[0105] Healthy 6-8 week old male C57BL / 6 mice, weighing 18-20 g, were purchased from the Experimental Animal Center of Air Force Medical University and maintained in an SPF animal room.

[0106] 2. Experimental Equipment

[0107] Small animal anesthesia machine, small animal nebulizer

[0108] 3. Experimental Reagents

[0109] Isoflurane, saline

[0110] 2. Experimental Methods

[0111] 1. 20 μL of saline solution was delivered to the lungs of C57BL / 6 mice by intranasal administration (after isoflurane anesthesia, an equal amount of drug solution was dripped into the left and right nasal cavities of mice at a uniform speed), exposed airway administration (after isoflurane anesthesia, the neck skin was cut to expose the airway, and the drug solution was injected after airway intubation), nebulization administration (the mice were placed in an induction box, the drug solution was added to the nebulizer cup, the power was turned on, and the corresponding administration time and concentration were set) and non-exposed airway instillation administration;

[0112] 2. Repeat the above operation after 1 week;

[0113] 3. Observe the survival status of mice in each group.

[0114] 3. Experimental Results

[0115] Using normal saline as a model drug, the drug was administered intranasally, through exposed airway administration, by nebulization, and by non-exposed airway instillation for 7 days, and then the drug was repeated once using the same administration method. The mice that were administered through non-exposed airway instillation had a good survival condition compared with the mice in the intranasal and nebulization groups, while the mice that were administered through exposed airway instillation all died within two days after repeated administration. Fig.18 shown.

[0116] In summary, the non-exposed airway instillation method provided by the present invention has significant comprehensive advantages over the currently commonly used lung tissue drug delivery technologies (intranasal administration, exposed airway administration, and atomization administration), and can achieve low-cost, non-invasive, and multiple mouse lung tissue-specific drug delivery of cell / non-cell drugs.

[0117] Example 2

[0118] like Figure 1-6 As shown, on the basis of Example 1, in order to reduce the probability of anesthesia leakage and reduce the impact of anesthesia on the experimenter, the supporting anesthesia device includes a base 1, a transparent tube 2, a top cover 3, etc. The base 1 is set as a cavity structure, and it is convenient for the experimenter to inject gaseous anesthetics. The transparent tube 2 is detachably arranged on the base 1, and the top cover 3 is detachably arranged on the top of the transparent tube 2. Therefore, the cavity structure of the transparent tube 2, the top cover 3 and the base 1 constitutes a sealed space. Under this arrangement, the anesthetic gas can flow from the cavity structure into the transparent tube 2, thereby anesthetizing the mice located in the transparent tube 2.

[0119] To form a cavity structure for the base 1, it includes a bottom cylinder 11 and a supporting net 12. The top of the bottom cylinder 11 is set as an opening, and an internal thread groove 114 is also provided on the upper side surface of the bottom cylinder 11. An annular plate 121 is provided on the outer side of the supporting net 12, and the annular plate 121 is threadedly installed at the internal thread groove 114. The supporting net 12 is threadedly installed on the bottom cylinder 11, which provides convenience for disassembling the supporting plate 12 and the bottom cylinder 11 according to requirements. Therefore, under the cooperation of the supporting net 12 and the bottom cylinder 11, a cavity structure is formed whose upper end can communicate with the transparent tube body 2 in terms of gas.

[0120] To be able to inject gaseous anesthetic into the cavity structure, a side tube 111 is communicatively provided on the side wall of the base 1. The side tube 111 is set as an L-shaped structure, and a rubber plug 112 is provided at the top. The needle body for injecting gaseous anesthetic is inserted into the rubber plug 112 to form a gas flow channel, so that the anesthetic drug can be injected into the cavity body under the action of an anesthesia machine, or the needle of a syringe is inserted into the rubber plug 112, and the excess anesthetic is drawn out under the action of the syringe, avoiding the problem of anesthetic leakage caused by the remaining anesthetic drug in the transparent tube body 2 after the mouse is anesthetized.

[0121] To stably set the transparent tube body 2 on the bottom cylinder 11 and provide convenience for disassembling the transparent tube body 2 and the bottom cylinder 11, an external thread groove 113 is provided on the upper side surface of the bottom cylinder 11. The external thread groove 113 is located outside the internal thread groove 114. A threaded structure 21 is provided at the bottom of the transparent tube body 2, and the bottom is inserted into the external thread groove 113. At the same time, a sealing ring is provided at the bottom of the external thread groove 113 to achieve the sealed connection between the transparent tube body 2 and the bottom cylinder 11.

[0122] To seal the top cover 3 on the transparent tube body 2, two circles of sealing rings 31 are provided on the inner and outer sides of the lower side surface of the top cover 3, and the two circles of sealing rings 31 are distributed on the inner and outer sides of the transparent tube body 2.

[0123] Embodiment 3

[0124] As Figure 7-14 shown, on the basis of Embodiment 1 or 2, to realize the hanging setting of the anesthetized mouse for drug injection, the supporting hanging device includes a base 1, a bracket 4, a hanging mechanism 6, a limiting plate group 5, etc. The base 1 has the same structure as the base 1 in the previous embodiment. The bracket 4 is set as a U-shaped structure and is stably installed on the base 1. In addition, it can be conveniently disassembled from the base 1. The hanging mechanism 6 is provided at the top of the bracket 4 and is connected to the upper front teeth of the mouse, and at the same time, the mouse is lifted to make it in a vertical state. To stably place the mouse, two groups of limiting plate groups 5 are provided, and they are respectively adjustably provided on the bracket 4, and the adjacent ends are in contact with the body of the mouse to limit the position of the mouse and make it stably placed.

[0125] In order to stably install the bracket 4 on the base 1, the bracket 4 includes a bottom ring plate 41, and the bottom ring plate 41 is threadedly installed on the bottom tube 11 of the base 1. In order to enhance the stability of the bracket 4, a mounting groove 13 is provided at the bottom of the bottom tube 11, and a pull-out plate 14 and a support plate 15 are provided at the mounting groove 13. The pull-out plate 14 is installed at the end of the support plate 15, and the pull-out plate 14 is installed movably with the cooperation of the support plate 15 and the mounting groove 13, so that the experimenter can control the pull-out plate 14 to protrude from the bottom tube 11, thereby enhancing the stability of the bottom tube 11 and the bracket 4. In order to stably install the pull-out plate 14 relative to the bottom tube 11, a rubber layer can be provided on the interface between the support plate 15 and the mounting groove 13.

[0126] In order to ensure stable installation of the limiting plate group 5 and the suspension mechanism 6, the bracket 4 further includes two vertical plates 42 and two U-shaped plates 44. The two vertical plates 42 are symmetrically and fixedly arranged above the bottom ring plate 41, and the two U-shaped plates 44 are respectively fixedly arranged on the top of the two vertical plates 42. The two limiting plate groups 5 are respectively sleeved on the two vertical plates 42, and the suspension mechanism 6 is installed on the two U-shaped plates 44.

[0127] In order to suspend the mouse through the suspension mechanism 6, the suspension mechanism 6 includes a suspension rope 61, a first end plate 62 and a second end plate 63. The first end plate 62 and the second end plate 63 are respectively installed on two U-shaped plates 44. One end of the suspension rope 61 is connected to the first end plate 62, and the other end is arranged through the second end plate 63, so as to realize the stable installation of the suspension rope 61 and provide support for changing the height of the mouse by adjusting the angle of the suspension rope 61 according to needs.

[0128] In order to ensure that the first end plate 62 and the second end plate 63 are stably installed on the U-shaped plate 44, the first end plate 62 and the second end plate 63 are both set to an I-shaped structure and are adapted to the U-shaped plate 44. Therefore, with the cooperation of the concave and convex structures adapted to the end plate and the U-shaped plate 44, the movement of the first end plate 62 and the second end plate 63 on the horizontal line is restricted, providing support for the suspension rope 61.

[0129] In order to ensure stable installation of the suspension rope 61, a conical through hole 621 is provided on the first end plate 62, and an end ball 611 is provided at the end where the suspension rope 61 is connected to the first end plate 62. The end ball 611 is located at the end of the through hole 621 with a larger size.

[0130] A frustum-shaped hole 632 is provided on the second end plate 63, and a support frame 634 is fixedly provided on the side away from the first end plate 62. The support frame 634 is arranged in a U-shaped structure, and a threaded rod 631 is threaded through the middle. A frustum-shaped column 635 is connected to the end of the threaded rod 631 through a bearing. The frustum-shaped column 635 can extend into the frustum-shaped hole 632. Since the suspension rope 61 passes through the frustum-shaped hole 632, after adjusting the height of the mouse, the threaded rod 631 can be rotated to control the frustum-shaped column 635 to insert into the frustum-shaped hole 632, squeezing the suspension rope 61 to keep a stable angle.

[0131] In addition, a convex column 633 is provided at the end of the support frame 634 protruding from the second end plate 63. A groove 45 is provided on the side wall of the U-shaped plate 44. The convex column 633 is arranged in the groove 45, thereby restricting the stable placement of the support frame 634 relative to the second end plate 63 and facilitating the rotation of the threaded rod 631.

[0132] In order to control the stable placement of the mouse after it is in a vertical state, the limiting plate group 5 includes a U-shaped plate 51, a first clamping plate 52, a second clamping plate 53 and an adjustable plate 54. The closed ends of the two U-shaped plates 51 are arranged adjacent to each other, and pressing arc plates 511 are provided at the ends close to each other, and they are respectively sleeved on the two vertical plates 42. The first clamping plate 52 and the second clamping plate 53 are connected by bolts and distributed on both sides of the vertical plate 42, and the bolts pass through the sliding holes 43 of the vertical plate 42. At the same time, the U-shaped plate 51 is adjustably connected to the first clamping plate 52. Therefore, with the cooperation of the first clamping plate 52 and the second clamping plate 53, the U-shaped plate 51 is stably installed relative to the bracket 4. The adjustable plate 54 is located on the side where the two vertical plates 42 are close to each other. A first magnet 512 and a second magnet 541 that repel each other are respectively provided on the adjacent sides of the pressing arc plate 511 and the adjustable plate 54. Under the action of the magnetic force, the two U-shaped plates 51 are forced to have a tendency to approach each other, thereby clamping the body of the mouse to make it stably placed.

[0133] In order to stably install the first clamping plate 52 and the second clamping plate 53 on both sides of the vertical plate 42, first plate bodies 521 and second plate bodies 531 with staggered distributions are provided on the sides of the first clamping plate 52 and the second clamping plate 53 close to each other, and the first plate bodies 521 and the second plate bodies 531 are both arranged in the sliding holes 43.

[0134] In order to stably and adjustably install the U-shaped plate 51 relative to the clamping plate, baffles 522 are fixedly arranged up and down on both vertical edges of the first clamping plate 52. The U-shaped plate 51 passes through the gap formed by the two baffles 522.

[0135] In order to be able to adjust the position of the adjustable plate 54 and to adapt to different needs, a limiting rod 543 is fixedly provided at one end of the adjustable plate 54, and an adjusting screw 542 is penetrated through the other end through a bearing connection. The adjusting screw 542 thread passes through the first clamping plate 52, and the limiting rod 543 passes through the first clamping plate 52. With the cooperation of the limiting rod 543 and the adjusting screw 542, the adjustable plate 54 is stably installed relative to the clamping plate, and the adjusting screw 542 is rotated to control the movement of the adjustable plate 54 to provide support.

[0136] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A non-exposed airway instillation method for lung-specific administration in mice, characterized in that: include Step 1: Prepare different types of drugs to be administered, namely hydrophilic drugs, hydrophobic drugs, and cell-based drugs; Step 2: placing the mouse in a matching anesthesia device and anesthetizing the mouse; then placing the anesthetized mouse on a matching hanging device to allow its body to droop naturally; Step 3: Use curved forceps to pull out the mouse's tongue until you feel resistance. This action can open the epiglottis and expose the airway. Step 4: Use a pipette to apply the drug to the root of the tongue and cover the mouse's nasal cavity with a gloved finger; at the same time, keep the mouse's tongue extended and the nasal cavity covered for 10 breaths to complete the drug administration.

2. The non-exposed airway instillation method for mouse lung-specific drug delivery according to claim 1, characterized in that: Hydrophilic drugs were dissolved in sterile saline and then sterilized by filtration with a 0.22 μm filter membrane; hydrophobic drugs were dispersed in sterile saline containing 30% corn oil and dissolved with ultrasound to form a suspension for later use; cell-based drugs were resuspended in serum-free culture medium in a clean bench for later use, and the concentration should be less than 10 5 / μL; the single administration volume of hydrophilic drugs, hydrophobic drugs, and cellular drugs should be less than 50μL, and the interval between multiple administrations should be greater than 6h.

3. A matching anesthesia device, used in the non-exposed airway instillation method of drug administration as claimed in claim 1, characterized in that: The invention comprises a base (1), wherein the base (1) comprises a bottom tube (11) and a support net (12), wherein the top of the bottom tube (11) is arranged to be open, and a side tube (111) is arranged on the side in communication therewith, and the support net (12) is detachably arranged at the opening of the bottom tube (11); a transparent tube body (2) is detachably and sealedly arranged above the base (1), and a top cover (3) is sealedly arranged on the top of the transparent tube body (2).

4. The anesthesia device according to claim 3, characterized in that: The side tube (111) is arranged in an L-shaped structure, and a rubber plug (112) is arranged on the top; an external thread groove (113) is arranged on the upper side of the bottom tube (11), a thread structure (21) is arranged on the bottom of the transparent tube body (2), and the bottom of the transparent tube body (2) is inserted into the external thread groove (113); an internal thread groove (114) is also arranged on the upper side of the bottom tube (11), and the internal thread groove (114) is located on the inner side of the external thread groove (113); an annular plate (121) is arranged on the outer side of the support net (12), and the annular plate (121) is threadedly installed at the internal thread groove (114); two circles of sealing rings (31) are arranged inside and outside the lower side of the top cover (3), and the two circles of the sealing rings (31) are distributed on the inner and outer sides of the transparent tube body (2).

5. A matching suspension device, used in the non-exposed airway instillation drug delivery method as claimed in claim 1, characterized in that: The invention comprises a base (1), a bracket (4) is detachably arranged on the base (1), two groups of limiting plate groups (5) are symmetrically arranged in the middle of the bracket (4), and a hanging mechanism (6) is arranged on the top of the bracket (4); the hanging mechanism (6) comprises a hanging rope (61), both ends of the hanging rope (61) are installed on the top of the bracket (4), and the middle part of the hanging rope (61) can be hung on the upper front teeth of a mouse; the adjacent ends of the two groups of limiting plate groups (5) can both be in contact with the body of the mouse.

6. A matching suspension device according to claim 5, characterized in that: The bracket (4) comprises a bottom ring plate (41), two vertical plates (42) and two U-shaped plates (44); the bottom ring plate (41) is threadedly mounted on the bottom tube (11) of the base (1); the two vertical plates (42) are symmetrically and fixedly arranged above the bottom ring plate (41); and the two U-shaped plates (44) are respectively fixedly arranged on the tops of the two vertical plates (42); a mounting groove (13) is arranged at the bottom of the bottom tube (11); a pull-out plate (14) and a support plate (15) are arranged at the mounting groove (13); the pull-out plate (14) is mounted at the end of the support plate (15), and the pull-out plate (14) can be extended out of the bottom tube (11).

7. A matching suspension device according to claim 5, characterized in that: The suspension mechanism (6) further includes a first end plate (62) and a second end plate (63). Both the first end plate (62) and the second end plate (63) are arranged in an I-shaped structure and are adapted to the U-shaped plate (44), and are installed on the U-shaped plate (44) at the same time. A through hole (621) with a conical structure is provided on the first end plate (62), and a frustum-shaped hole (632) is provided on the second end plate (63). The suspension rope (61) passes through the frustum-shaped hole (632), and the end of the suspension rope (61) extends into the through hole (621). At the same time, the end ball (611) installed at the end of the suspension rope (61) is located at the end with a larger size of the through hole (621).

8. A matching suspension device according to claim 7, characterized in that: A support frame (634) is fixedly provided on the side of the second end plate (63) away from the first end plate (62). The support frame (634) is arranged in a C-shaped structure, and a threaded rod (631) is provided through the middle in a threaded manner. A frustum-shaped column (635) is provided at the end of the threaded rod (631). The frustum-shaped column (635) can extend into the frustum-shaped hole (632) to squeeze the suspension rope (61). A convex column (633) is provided at the end of the support frame (634) protruding from the second end plate (63). A groove (45) is provided on the side wall of the U-shaped plate (44). The convex column (633) is arranged in the groove (45).

9. A matching suspension device according to claim 5, characterized in that: The limiting plate group (5) includes a C-shaped plate (51), a first clamping plate (52), a second clamping plate (53) and an adjustable plate (54). The first clamping plate (52) and the second clamping plate (53) are connected by bolts and are distributed on both sides of the vertical plate (42), and the bolts pass through the sliding holes (43) of the vertical plate (42). The adjustable plate (54) is located on the side where the two vertical plates (42) are close to each other. The vertical plate (42) passes through the C-shaped plate (51), and a pressing arc plate (511) is fixedly provided at the end of the vertical plate (42). A first magnet (512) and a second magnet (541) that repel each other are respectively provided on the adjacent sides of the pressing arc plate (511) and the adjustable plate (54).

10. A matching suspension device according to claim 9, characterized in that: On the side where the first clamping plate (52) and the second clamping plate (53) are close to each other, a first plate body (521) and a second plate body (531) are arranged in a staggered manner, and both the first plate body (521) and the second plate body (531) are arranged in the sliding hole (43). On both vertical edges of the first clamping plate (52), baffles (522) are fixedly provided up and down. The C-shaped plate (51) passes through the gap formed by the two baffles (522). A limiting rod (543) is fixedly provided at one end of the adjustable plate (54), and an adjusting screw (542) is connected through the other end by a bearing and is threaded through the first clamping plate (52). The limiting rod (543) passes through the first clamping plate (52).

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