Construction method and application of acute radioolfaction disorder animal model

Abstract

The invention discloses a construction method and application of an acute radioactive olfactory disorder animal model, a linear accelerator is adopted to perform single dose gradient external irradiation on the head and neck of a mouse, and the model can stably simulate dose-related acute radioactive olfactory disorder and has the advantages of high survival rate, low cost, simplicity and convenience in operation and good repeatability. And a foundation is laid for related researches on an acute radioolfaction disorder generation mechanism and prevention and treatment of head and neck malignant tumor radiotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical and radiomedical research technology, specifically relating to a method for constructing an animal model of acute radiation-induced olfactory dysfunction, and the application of this animal model in research related to acute radiation-induced olfactory dysfunction in radiotherapy for head and neck malignant tumors. Background Technology

[0002] The sense of smell is an important chemoreceptor system in the human body, playing an irreplaceable role in maintaining appetite, perceiving environmental danger signals, and emotional memory. Acute olfactory impairment following radiotherapy is common in patients with head and neck malignancies, and is often overlooked by both patients and researchers due to the significant variability in clinical symptoms. Existing research indicates that the imbalance between regenerating and apoptotic cells caused by 4 Gy radiation to the nose in mice is one of the mechanisms of olfactory dysfunction; the maximum success rate of olfactory modeling after 8 Gy radiation (28 days after modeling) is only 25%, with unclear olfactory dysfunction symptoms and a low modeling success rate. Currently, no research systematically explores the acute evolution of radiation-induced olfactory impairment at the behavioral, histological, and molecular levels. Summary of the Invention

[0003] To address the lack of stable, reproducible animal models that accurately reflect the pathological process of acute radiation-induced olfactory dysfunction in existing technologies, this invention provides a method for constructing an animal model of acute radiation-induced olfactory dysfunction. This method can be used to study the pathogenesis of radiation-related olfactory damage and provide an experimental basis for the development of related prevention and treatment strategies.

[0004] The technical solution of this invention is: a method for constructing an animal model of acute radiation-induced olfactory dysfunction, comprising the following steps: (1) Preparation of experimental animals: Healthy male 8-week-old BALB / c mice were selected and acclimatized for 7 days in a standard environment with free access to water and food. (2) Radiation modeling: Experimental mice were randomly selected into at least one model group. After weighing and anesthesia, the mice were arranged in a supine position with their heads fixed. A single head and neck radiation was performed using a linear accelerator with a radiation dose gradient of 8 Gy to 24 Gy. The radiation field was from the tip of the nose to the neck. The number of days for radiation modeling was recorded as 0. (3) Model evaluation: At different time points after modeling, the establishment of the acute radiation olfactory disorder animal model was comprehensively evaluated by observing the general condition of each group of mice, olfactory function test, morphology of olfactory bulb and olfactory epithelium, and expression level of olfactory epithelial aquaporin 3. The general condition included mental and activity status, fur condition, eating and drinking status, stool and odor, and whether there was any other discomfort. Body weight was also recorded.

[0005] Preferably, in step (2), the single radiation dose is 8 Gy - 16 Gy.

[0006] Ideally, in step (2), the single radiation dose is 12 Gy.

[0007] Preferably, in step (3), the olfactory function test includes the following steps: restricting food intake for 24 hours before the test, and allowing free access to water; laying a 2 cm thick layer of bedding material in the test cage, and randomly burying food balls 0.5 cm away from the surface; placing mice one by one, and recording the time from when the mouse enters the test cage to when it grabs the food ball with its front paws and nibbles on it; if the mouse still cannot find the food ball after 5 minutes, it is considered to have olfactory impairment. Each mouse is tested 3 times at intervals, and the average value is taken.

[0008] Preferably, the morphological observation of the olfactory bulb and olfactory epithelium in step (3) includes the following steps: The morphological observation of the olfactory bulb and olfactory epithelium included the following steps: After weighing, anesthesia was performed by intraperitoneal injection of 0.25% tribromoethanol at a dose of 0.5 ml / 20 g; at least 20 ml of 0.9% saline was perfused into the left ventricle; then, nasal tissue was excised 0.5 cm posterior to the tip of the nose, nasal skin and soft tissue were removed, the structures on both sides were sharply dissected, and the tissue was embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and scanned. The overall morphology of the olfactory bulb and its layers were observed using Case Viewer 2.3, and the changes in the layers of the olfactory epithelium were observed and compared. The thickness of the olfactory epithelium and the thickness of the supporting cells were measured vertically at three identical locations, and the average values ​​were recorded.

[0009] Preferably, the olfactory epithelial aquaporin 3 expression level analysis in step (3) includes the following steps: After weighing, anesthesia was administered via intraperitoneal injection of 0.25% tribromoethanol at a dose of 0.5 ml / 20 g. At least 20 ml of 0.9% saline was perfused into the left ventricle. Nasal tissue was then excised 0.5 cm from the tip of the nose, and the nasal skin and soft tissue were removed. The structures on both sides were sharply dissected, embedded in paraffin, sectioned, and stained with aquaporin 3 immunohistochemically. The sections were then scanned. The following calculations were performed: ① Immunological response score (IRS) = SI × PP, where SI is the positive intensity and PP is the percentage of positive cells; SI is divided into 3 grades: grade 0 no positive staining, grade 1 pale yellow weak positive, grade 2 brownish-yellow moderate positive, and grade 3 brownish-red strong positive; positive cell percentage = number of positive cells / total number of cells; PP is divided into 4 grades: grade 0 0-5%, grade 1 6%-25%, grade 2 26%-50%, grade 3 51%-75%, and grade 4 >75%; ② Histochemical score of the olfactory epithelial region (H-Score) = ∑ (pi × i) = (percentage of weak intensity cells × 1) + (percentage of moderate intensity cells × 2) + (percentage of strong intensity cells × 3), where i represents the staining intensity score: negative (no staining) = 0 points; weak positive (pale yellow) = 1 point; moderate positive (brownish-yellow) = 2 points; strong positive (brownish-red) = 3 points; pi represents the percentage of positive cells at each intensity level.

[0010] Preferably, in step (3), the mental and activity status, fur condition, food and water intake, stool and odor, and any other discomfort of each group of mice are observed starting from day 1 of modeling, and body weight is recorded; olfactory function tests are performed on mice at five time points: day 2, day 4, week 1, week 2, and week 4 of modeling; one mouse is randomly selected at each of day 2, day 4, week 1, week 2, and week 4 of modeling for observation of olfactory bulb and olfactory epithelium morphology; and one mouse is randomly selected at each of day 2, day 4, week 1, week 2, and week 4 of modeling for analysis of olfactory epithelial aquaporin 3 expression level.

[0011] An animal model of acute radiation-induced olfactory dysfunction was also provided for use in research related to radiotherapy for head and neck malignancies.

[0012] The beneficial technical effects of the present invention are as follows: (1) Precise dose optimization: Through dose gradient experiments, the effective modeling range of 8Gy-16Gy was determined, and 12Gy was determined as the optimal modeling dose, which can effectively induce damage while ensuring animal survival rate and model stability.

[0013] (2) Multidimensional comprehensive evaluation system: Combining general conditions, olfactory function test, morphology of olfactory bulb and olfactory epithelium, and expression level of olfactory epithelial aquaporin 3, the model is comprehensively and objectively verified to ensure that the model can truly reflect the pathophysiological changes of clinical acute radiation injury.

[0014] (3) Repeatability and operability: The method has clear steps, controllable conditions, is easy to standardize, and has high repeatability.

[0015] (4) Clear application objectives: The constructed model is specifically used for scientific research for purposes other than disease diagnosis and treatment, such as research on complications and prevention of radiotherapy for head and neck malignant tumors. Attached Figure Description

[0016] Figure 1 Displayed as a radial model. Figure 1 A: Mouse radiation positioning and radiation field; Figure 1 B: Linear accelerator 6 MV X-ray, 600 c Gy / min, isocentric 100 cm different dose radiation panoramic view of mouse head and neck.

[0017] Figure 2 The image shows HE staining (40×) and the thickness of the olfactory epithelium and supporting cell layer. Figure 2 AF represents the olfactory epithelium HE staining of the blank control group, 8Gy, 12Gy, 16Gy, 20Gy, and 24Gy model groups on Day 4 of modeling; Figure 2 G: Changes in the thickness of the olfactory epithelium; Figure 2 H: Supports changes in cell layer thickness. *P<0.05, **P<0.01.

[0018] Figure 3 The olfactory bulb structure is shown as HE-stained (10×) on Day 4 of modeling. Figure 3 A is the control group; Figure 3 B represents the 20Gy model group; Figure 3 C represents the 24 Gy model group. It includes the NFL olfactory nerve fiber layer, GL synaptic glomeruli layer, EPL outer plexiform layer, ML mitral cell layer, IPL inner plexiform layer, and GCL granule cell layer.

[0019] Figure 4 The image shows olfactory epithelium AQP-3 immunohistochemical staining (40×). Figure 4 A represents the blank control group; Figure 4 B is an 8Gy module; Figure 4 C is a 12Gy module; Figure 4 D is a 16Gy module; Figure 4 E is a 16Gy module; Figure 4 F is a 20Gy module; Figure 4G represents AQP-3 expression in the olfactory epithelium during the modeling week 1, **P < 0.01. Detailed Implementation

[0020] The method for constructing this acute radiation-induced olfactory dysfunction animal model includes the following steps: (1) Preparation of experimental animals: Several healthy male 8-week-old BALB / c mice were selected and acclimatized for 7 days in a standard environment with free access to water and food. (2) Grouping and radiation modeling: Mice were randomly divided into a control group and ≥1 model group. The model group mice were anesthetized by intraperitoneal injection according to their weight, and after being fixed in a supine parallel position, a linear accelerator was used to irradiate their head and neck (radiation field from the tip of the nose to the neck) with a single dose between 8 Gy and 24 Gy, with the preferred dose being 8 Gy to 16 Gy and the optimal dose being 12 Gy (the model mice can stably show typical acute radiation injury symptoms, and the mortality rate is low, and the model has the best repeatability). (3) Model evaluation: Starting from day 1 of modeling, observe the mental and activity status, fur condition, food and water intake, stool and odor, and any other discomfort of each group of mice, and record body weight; conduct the following evaluations at five time points: day 2, day 4, week 1, week 2, and week 4 of modeling: ① olfactory function test, ② olfactory bulb and olfactory epithelium morphology, ③ olfactory epithelial aquaporin 3 expression level.

[0021] This invention helps to deepen our understanding of the pathogenesis of acute radiation-induced olfactory dysfunction. It is applicable to research on the mechanism of radiation-induced olfactory dysfunction, verification of treatment plans, and rehabilitation of olfactory function, and has high practicality and promising prospects for promotion.

[0022] Preferably, in step (2), a linear accelerator with 6MV X-rays and 600 c Gy / min is used, with a single radiation dose of 100cm at the isocenter. The preferred radiation dose is 8Gy to 16Gy, and the optimal dose is 12Gy.

[0023] Preferably, in step (3①), the mice are restricted from eating for 24 hours before the test, but are allowed free access to water; a 2cm thick layer of bedding is laid in the test cage, and food balls are randomly buried 0.5cm from the surface; mice are placed in one by one, and the time from when the mouse enters the test cage to when it grabs the food ball with its front paw and nibbles on it is recorded; if the mouse still cannot find the food ball after 5 minutes, it is considered to have olfactory dysfunction. Each mouse is tested 3 times at intervals, and the average value is taken.

[0024] Preferably, in step (3②), after weighing the mice, they are anesthetized by intraperitoneal injection of 0.25% tribromoethanol at a dose of 0.5 ml / 20 g; at least 20 ml of 0.9% saline is perfused into the left ventricle; then, nasal tissue is cut from 0.5 cm behind the tip of the nose, the nasal skin and soft tissue are removed, the structures on both sides are sharply separated, the tissue is embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and scanned. The overall morphology and layers of the olfactory bulb are observed using Case Viewer 2.3, the changes in the layers of the olfactory epithelium are observed and compared, and the thickness of the olfactory epithelium and the thickness of the supporting cells are measured vertically at three identical locations, and the average value is recorded.

[0025] Preferably, in step (3③), after weighing the mice, they are anesthetized by intraperitoneal injection of 0.25% tribromoethanol at a dose of 0.5 ml / 20 g; at least 20 ml of 0.9% saline is perfused into the left ventricle; then, nasal tissue is cut from 0.5 cm behind the tip of the nose, the nasal skin and soft tissue are removed, the structures on both sides are sharply separated, and the tissue is embedded in paraffin, sectioned, stained with aquaporin 3 immunohistochemically, and scanned. The following calculations were performed: ① Immunological response score (IRS) = SI × PP, where SI is the positive intensity and PP is the percentage of positive cells; SI is divided into 3 grades: grade 0 no positive staining, grade 1 pale yellow weak positive, grade 2 brownish-yellow moderate positive, and grade 3 brownish-red strong positive; positive cell percentage = number of positive cells / total number of cells; PP is divided into 4 grades: grade 0 0-5%, grade 1 6%-25%, grade 2 26%-50%, grade 3 51%-75%, and grade 4 >75%; ② Histochemical score of the olfactory epithelial region (H-Score) = ∑ (pi × i) = (percentage of weak intensity cells × 1) + (percentage of moderate intensity cells × 2) + (percentage of strong intensity cells × 3), where i represents the staining intensity score: negative (no staining), 0 points; weak positive (pale yellow), 1 point; moderate positive (brownish-yellow), 2 points; strong positive (brownish-red), 3 points; pi represents the percentage of positive cells at each intensity level.

[0026] Through dose gradient experiments, the effective modeling range of 8 Gy-16 Gy was identified, and 12 Gy was determined as the optimal modeling dose. This dose effectively induced damage while ensuring animal survival, demonstrating high model stability. The methodology is clear, the conditions are controllable, it is easy to standardize, and it exhibits high reproducibility. The constructed model is specifically designed for scientific research aimed at non-disease diagnosis and treatment, such as research on complications and prevention related to radiotherapy for head and neck malignancies.

[0027] Preferably, radiation injury causes disorders in the aqueous metabolism of the olfactory epithelium, and radiation-induced nasal and sinus inflammation leads to conductive olfactory disorders. The mechanism of sensory neural olfactory disorder is the occurrence mechanism of olfactory injury after radiation.

[0028] Preferably, radiation upregulates the expression of AQP-3 in the olfactory epithelium of the nasal mucosa.

[0029] Preferably, the single radiation dose is between 8 Gy and 16 Gy.

[0030] Optimally, the single radiation dose is 12 Gy.

[0031] The following will give a detailed description of the embodiments of the present invention.

[0032] This study is based on the scientific hypothesis that radiation disrupts the secretion of glands in the olfactory epithelium and breaks the balance of the surface microenvironment, establishes an animal model of acute radiation-induced olfactory disorder, and explores the related mechanisms.

[0033] 1. Materials and Methods 1.1 Experimental Animals BALB / c mice are widely used and are very sensitive to radiation. Single male mice can reduce the inter-group differences and the protective effect of estrogen levels in the body on olfactory sensory neurons (OSNs). Thirty BALB / c male 8-week-old mice (animal license number: SCXK (Beijing) 2019-0010) with a body weight of (20±2) g were selected for the study. Before the experiment, the mice were adaptively fed in a standard environment for 7 days with free access to water and food.

[0034] 1.2 Modeling Method The mice were randomly divided into six groups: 5 mice in the control group (0 Gy), 5 mice in each of the low-dose model groups (8 Gy group, 12 Gy group), and 5 mice in each of the high-dose model groups (16 Gy group, 20 Gy group, 24 Gy group). After weighing, the mice were anesthetized by intraperitoneal injection of 0.25% tribromoethanol at a dose of 0.5 ml / 20 g, placed in a supine position in parallel, and the heads of the mice were fixed. A linear accelerator (6 MV X-ray, 600 cGy / min) was used to irradiate the head and neck of the mice with a multi-dose (8 Gy, 12 Gy, 16 Gy, 20 Gy, 24 Gy) single dose at an isocenter of 100 cm (the radiation field was from the tip of the nose to the neck), see Figure 1 . After the radiation, the anesthesia was restored, and the number of days of radiation modeling was recorded as Day 0.

[0035] 1.3 Evaluation Indexes and Methods 1.3.1 General Conditions Starting from Day 1 of modeling, the mental state, activity status, fur condition, food and water intake, feces and urine traits and odors, and other discomforts of the mice in each group were observed, and the body weight was recorded.

[0036] 1.3.2 Experiment with buried food pellets Behavioral observations were conducted on six groups of mice at five time points: Day 2, Day 4, Week 1, Week 2, and Week 4. Food intake was restricted for 24 hours prior to testing (<0.2 g / day / mouse), but water was allowed free access. A 2 cm thick layer of bedding was placed in the test cage, with food balls randomly buried 0.5 cm below the surface. Mice were placed in the cage one by one, and the time from entering the cage to the mouse grasping and nibbling on the food ball with its forepaws was recorded (smelling the ball was not counted). If the mouse could not find the ball within 5 minutes, it was considered to have olfactory dysfunction. Each mouse was tested three times at intervals, and the average value was taken.

[0037] 1.3.3 Sampling and Specimen Processing One animal was randomly sacrificed on Day 2, Day 4, Week 1, Week 2, and Week 4 of the model group. After weighing, the animals were anesthetized by intraperitoneal injection of 0.25% tribromoethanol (0.5 ml / 20 g), perfused with 20 ml of 0.9% saline via the left ventricle, decapitated, and after the skin and adipose tissue were removed, the animals were placed in 4% paraformaldehyde fixative and fixed at 4°C for 24 h. The animals were then decalcified with 0.5M EDTA for 14 days, with the decalcification solution changed every 2-3 days. After paraffin embedding, sectioning, and HE staining, the sections were scanned and examined using Case Viewer 2.3 to observe the overall morphology and layers of the olfactory bulb. Changes in the layers of the olfactory epithelium (including the olfactory epithelium, lamina propria, supporting cells, olfactory sensory neurons, basal cells, nerve bundles, and Bowman's glands) were observed and compared. The thickness of the olfactory epithelium and supporting cells were measured vertically at three identical locations, and the average values ​​were recorded.

[0038] Immunohistochemical staining was performed using Anti-Aquaporin 3 Rabbit pAb (Servicebio Cat# GB11395-100) and Aipathwell was used. ® (Servicebio ®The expression of aquaporin-3 (AQP-3) in the olfactory epithelium and olfactory bulb was qualitatively analyzed using digital pathological image analysis software. IRS = SI (positive intensity) × PP (positive cell ratio): SI is divided into 3 grades, grade 0 is no positive staining, grade 1 is pale yellow and weakly positive, grade 2 is brownish-yellow and moderately positive, and grade 3 is brownish-red and strongly positive; positive cell ratio = number of positive cells / total number of cells. PP can be divided into 4 grades, grade 0 is 0-5%, grade 1 is 6%-25%, grade 2 is 26%-50%, grade 3 is 51%-75%, and grade 4 is >75%. The olfactory epithelial region is semi-quantitatively calculated using the Histochemistry score (H-score). The formula is: H-Score = ∑(pi × i) = (percentage of weak intensity cells × 1) + (percentage of moderate intensity cells × 2) + (percentage of strong intensity cells × 3), where i represents the grade of positive cells: negative (no staining), 0 points; weakly positive (pale yellow), 1 point; moderately positive (brownish-yellow), 2 points; strongly positive (brownish-brown), 3 points. pi represents the percentage of positive cells in the corresponding grade. The H-score is a value between 0 and 300. Higher IRS and H-score values ​​indicate a stronger overall positive intensity, considering both the depth and quantity of positivity.

[0039] 1.4 Statistical Analysis Data were analyzed and processed using Graph Pad Prism 9. Quantitative data were expressed as mean ± standard deviation (x ± s). Independent samples t-tests were used to compare two groups, provided they were normally distributed and homogeneous in variance. One-way ANOVA was used for comparisons among multiple groups, and the least significant difference (LSD) test was used for pairwise comparisons between groups. If the conditions were not met, nonparametric tests were used. A p-value < 0.05 was considered statistically significant.

[0040] 2. Results 2.1 General Case Before radiation, mice in all groups were active, had good appetites, and healthy, glossy fur. On Day 4 of modeling, some mice in the model groups began to show signs of lethargy. On Week 1 of modeling, mice in the low-dose group were listless and less active, with no obvious abnormalities in fur or urination / defecation. The high-dose group showed the most severe symptoms, including lethargy, immobility, disheveled fur, arched back, unilateral or bilateral eye closure, and foul-smelling feces. From 9 to 10 days, four mice in the 20 Gy and 24 Gy groups gradually developed slowed movement and neck stiffness. Two weeks after modeling, the mental and activity levels of the low-dose group gradually returned to normal.

[0041] On Day 2 of the modeling process, the low-dose group showed a significant difference in body weight compared to the control group (P < 0.05); on Week 1 of the modeling process, the high-dose group showed a significant decrease in body weight compared to the low-dose group (P < 0.0001). During the experiment, one mouse in the 20 Gy group developed purulent discharge from its left eye, and one mouse developed lip atrophy; in the high-dose group, 1, 2, and 3 mice died 9-11 days after modeling, respectively.

[0042] 2.2 Experiment with buried food pellets After modeling, on Day 2, the high-dose group showed a significantly longer ball-finding time compared to the control group (P < 0.001) and the low-dose group (P < 0.005), while there was no significant difference between the low-dose group and the control group. On Day 4, both model groups showed a significantly longer ball-finding time compared to the control group (P < 0.05). In Week 1, 6 / 9 of the mice in the high-dose group experienced olfactory dysfunction, with no statistically significant difference between the two model groups. Olfactory function recovered somewhat in Week 2 after modeling.

[0043] 2.3 Histopathology 2.3.1 HE staining 2.3.1.1 Olfactory epithelium After tissue sections were stained with hematoxylin and eosin (HE), they were observed under a microscope. Figure 2-3 In mice, the olfactory epithelium is widely distributed, and its location varies. The olfactory epithelium is a pseudostratified ciliated columnar epithelium, with all basal cells attached to the basement membrane. Due to the varying cell heights, the nuclei are not located on the same horizontal plane, resulting in a "multi-layered nucleus" appearance under the microscope. Figure 2 AF, representing the olfactory epithelium at approximately the same location (40×) in mice from the blank control group, and the 8 Gy, 12 Gy, 16 Gy, 20 Gy, and 24 Gy model groups, respectively, showed the following qualitative analysis: ① the supporting cell layer thinned after radiation modeling; ② the number of olfactory epithelial layers decreased. Quantitative measurements were taken at three locations, and the average value was calculated. Figure 2 GH. The olfactory epithelium and supporting cell layer in the high-dose group were significantly thinner than the blank control group on Day 2 of modeling (P<0.05). On Day 4 of modeling, the thickness of the olfactory epithelium in both model groups was significantly reduced compared with the blank control group, with the high-dose group showing the most significant reduction (P<0.01). The supporting cell layer in both model groups was significantly thinner than the blank control group (P<0.05). There was no significant difference between the two model groups.

[0044] 2.3.1.2 Olfactory bulb Figure 3 A and B are olfactory bulbs at approximately the same location (10×) in the blank control group, the 20Gy model group, and the 24Gy model group, respectively. Qualitative analysis shows that the number of synaptic globules, outer plexiform layer, and monk's cap cell layer is significantly reduced and thinned after radiation modeling.

[0045] 2.3.2 Immunohistochemical staining After immunohistochemical staining of tissue sections, as shown Figure 4 In both the blank control group and the high-dose group, the olfactory bulb IRS was 0, indicating that AQP-3 was barely expressed in the olfactory bulb. The olfactory epithelial IRS in the blank control group was 4, while that in the model group was 6. Qualitative analysis showed that AQP-3 expression in the olfactory epithelium was upregulated after radiation modeling in mice, particularly in the outer layer of Bowman's glands (i.e., the basolateral membrane). Combined with H-score changes, the depth and number of positive cells increased with increasing radiation dose. Semi-quantitative analysis of AQP-3 H-score in the olfactory epithelial region was performed in Week 1 of the modeling period. Figure 4 G), the expression in the low-dose group and the high-dose group was significantly different from that in the control group (P<0.01), while there was no significant difference between the two model groups.

[0046] 3. Discussion This study induced olfactory impairment in male BALB / c mice through single external irradiation at different radiation dose gradients, evaluating olfactory function, tissue structure, and molecular expression from multiple dimensions. Clinically, radiotherapy-induced olfactory damage accounts for 7%-76% of cases, with some heterogeneity in onset and spontaneous recovery. The olfactory system comprises the olfactory epithelium, olfactory bulb, and olfactory center. Odor perception begins with odor molecules binding to odor receptors in olfactory sensory neurons (OSNs) within the cilia of the main olfactory epithelium (MOE) in the nasal cavity. These receptors then project to the glomerulus within the olfactory bulb (OB). The axonal terminals of the OSNs in the glomerulus synapse with the excitatory projection neurons of the OB—monk's cells and mitral / tufted cells (M / T)—further projecting to the olfactory cortex. During the process of odor molecules reaching the olfactory epithelial mucosa, the pathological state of the nasal cavity and sinuses can affect the binding of odor molecules to olfactory receptors. For example, nasal and sinus inflammation or masses can lead to conductive olfactory dysfunction. The nasal mucosa contains abundant glands and goblet cells, producing a large amount of secretions to form a mucus blanket on the mucosal surface that moves with the movement of cilia, also known as airway surface liquid (ASL). This ASL contains mucin and 95%-97% water. Aquaporins in the olfactory epithelium of the nasal mucosa can regulate the composition, thickness, and mucociliary movement rate of ASL by controlling water transmembrane transport; therefore, their expression levels can reflect changes in pathological conditions. Studies have shown that in unilateral ostomy (OB), there is a mutually inhibitory connection circuit between the inner and outer olfactory glomeruli, mainly mediated by external tufted cells (ETCs) and granule cells (GCs). This circuit can enhance the receptive field of M / T cells to weak signals, participate in the formation of inhibitory effects on adjacent olfactory glomeruli, and enhance the difference in olfactory information.

[0047] At the olfactory function level, mice in the high-dose model group showed olfactory decline as early as Day 2 of modeling, while mice in the low-dose model group showed olfactory decline starting on Day 4 of modeling. While there was some self-repair ability over time, it could not fully recover to pre-radiation levels. This result is consistent with clinical reports and effectively simulates the process of acute radiation-related olfactory dysfunction. At the tissue level, the number of olfactory epithelial, olfactory bulb synaptic bulb layer, outer plexiform layer, and mitral cell layer was significantly reduced in the model group mice. The radiation-induced olfactory dysfunction may be related to radiation damage disrupting the structure of the olfactory epithelium and olfactory bulb, and the excitation-inhibition homeostasis of the conduction circuit: ① Reduced number of olfactory epithelial layers, ciliary loss, disordered cilia movement, and insufficient odor receptor binding sites; ② Reduced ETCs in the outer plexiform layer weakened the inhibitory connection of the olfactory bulb circuit, reducing the difference in odor information input between the two nostrils; ③ Reduced number of gated M / T cells, which are olfactory input, prevented olfactory nerve stimulation from being transmitted to the olfactory cortex. Olfactory epithelial cells are metabolically active and proliferate rapidly, with OSNs constantly regenerating and renewing. However, due to the disruption of the balance between excitation and inhibition in the neural circuits within the olfactory bulb, olfactory function cannot return to its initial state before radiation exposure. After radiation exposure, the expression of AQP-3 in the olfactory epithelium of the nasal mucosa is upregulated, possibly due to increased secretory activity of the nasal Bowman's glands, altering the distribution of water within the nose, inducing mucus blanket dysfunction, and leading to nasal and sinus inflammation. Odor molecules cannot bind normally to olfactory receptors, resulting in conductive olfactory dysfunction.

[0048] 4. Conclusion This study validated that radiation injury leads to metabolic disorders of olfactory epithelium fluid and found that, in addition to conductive olfactory impairment caused by radiation-induced nasal and sinus inflammation, sensory nerve olfactory impairment may be the main mechanism of olfactory damage after radiation. In this study, radiation doses exceeding 16 Gy significantly increased the mortality rate in mice, and a single 12 Gy radiation dose to the head and neck was suitable for establishing an animal model of radiation-induced olfactory impairment in terms of olfactory function and histopathology.

[0049] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of acute radiation-induced olfactory dysfunction, characterized by: It comprises the following steps: (1) Select healthy BALB / c male 8-week-old mice, adaptively feed in standard environment for 7 days, free drinking water and food; (2) Randomly select experimental mice into at least one model group, weigh after anesthesia, supine position parallel arrangement, and fix the mouse head, apply linear accelerator, single head and neck radiation, radiation dose gradient is 8Gy-24Gy, radiation field from the nose to the neck, record the radiation modeling days as 0; (3) At different time points after modeling, the general state of each group of mice, olfactory function test, olfactory bulb and olfactory epithelium morphology, olfactory epithelium aquaporin 3 expression level were observed, and the establishment of acute radiation-induced olfactory dysfunction animal model was comprehensively evaluated. General state includes: mental and activity condition, fur state, food and water intake, stool odor, and other discomforts, and records the body weight.

2. The method of claim 1, wherein the acute radiation-induced anosmia animal model is constructed by: In step (2), the single radiation dose is 8Gy-16Gy.

3. The method of claim 2, wherein the acute radiation-induced anosmia animal model is constructed by: In step (2), the single radiation dose is 12Gy.

4. The method of claim 1, wherein the acute radiation olosmia animal model is constructed by: In step (3), the olfactory function test includes the following steps: limit food intake 24h before testing, free drinking water; 2cm thick bedding is laid in the test cage, and food balls are randomly buried at a distance of 0.5cm from the surface; put in the mouse one by one, record the time from the mouse entering the test cage to holding the food ball with the front paw and nibbling; if the ball is not found after 5 minutes, it is recorded as olfactory dysfunction; each mouse is tested 3 times with an interval of 3 minutes, and the average value is taken.

5. The method of claim 1, wherein the acute radiation olosmia animal model is constructed by: In step (3), the olfactory bulb and olfactory epithelium morphology observation includes the following steps: after weighing, 0.25% tri bromoethanol is injected intraperitoneally at 0.5ml / 20g for anesthesia; perfuse at least 20ml of 0.9% normal saline through the left ventricle, then cut the nose tissue 0.5cm behind the nose tip, remove the nose skin and soft tissue, sharply separate the bilateral structure, paraffin embedding, sectioning, HE staining, scanning the section, applying Case Viewer 2.3 to observe the overall morphology of the olfactory bulb and each layer, observe and compare the changes of each layer of the olfactory epithelium, and measure the thickness of the olfactory epithelium and the thickness of the supporting cells at three same positions vertically, and record the average value.

6. The method of claim 1, wherein: The step (3) of observing the expression level of the olfactory epithelium aquaporin 3 includes the following steps: after weighing, 0.25% triosmium ethanol is injected into the abdominal cavity at 0.5 ml / 20 g for anesthesia; 0.9% normal saline is perfused through the left ventricle for at least 20 ml, and then the nasal tissue is cut from the back 0.5 cm of the nose tip, the nasal skin and soft tissue are removed, the two sides are sharply separated, paraffin embedding, sectioning, aquaporin 3 immunohistochemical staining, and scanning of the section are performed; and calculation and analysis are performed on ①immunoreaction integral IRS=SI×PP, SI is positive intensity, and PP is positive cell ratio; SI is divided into three levels, 0 level has no positive color, 1 level has weak yellow weak positive, 2 level has brownish yellow moderate positive, and 3 level has strong brown strong positive; the positive cell ratio=positive cell number / total cell number, and PP is divided into four levels, 0 level is 0-5%, 1 level is 6%-25%, 2 level is 26%-50%, 3 level is 51%-75%, and 4 level is >75%; ②histochemical score H-Score of the olfactory epithelium region =∑(pi×i)=(weak positive cell percentage ×1) + (moderate positive cell percentage ×2) +(strong positive cell percentage ×3), wherein i represents the integral of staining intensity: negative without color, 0 points; weak positive light yellow, 1 point; moderate positive brownish yellow, 2 points; strong positive brown, 3 points; and pi represents the positive cell percentage of each intensity level.

7. Use of an acute radioodorous animal model for purposes other than disease diagnosis and treatment, characterized in that: The application includes: using the acute radiation olfactory disorder animal model obtained by the construction method of any one of claims 1-6 in the research of acute radiation olfactory disorder related to radiotherapy of head and neck malignant tumors.

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