Method for establishing a chronic sinusitis model in non-human primates

Abstract

The present invention relates to the technical field of disease model construction, and more specifically, to a method for establishing a model of chronic sinusitis in non-human primates. The method comprises the step of placing an expandable sponge in the middle nasal passage of the non-human primate. The method does not require special instruments or equipment and has low cost. The construction of the present invention successfully establishes a chronic sinusitis model in non-human primates for the first time, laying a foundation for exploring the occurrence and development of sinusitis, filling a technical gap in existing sinusitis model construction, and having significant clinical and economic value.

Description

Technical Field

[0001] The present invention relates to the technical field of disease model construction, and in particular to a method for establishing a model of chronic sinusitis in non-human primates. Background Art

[0002] Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinus and nasal mucosa, with a course of more than 12 weeks. It is one of the most common inflammatory respiratory diseases, with an incidence of 5-28% in the general population. Clinically, it can be divided into two types: (1) chronic rhinosinusitis without nasal polyps (CRSsNP); and (2) chronic rhinosinusitis with nasal polyps (CRSwNP). Ventilation and drainage dysfunction caused by anatomical abnormalities in the ostiomeatal complex is generally considered to be the pathogenesis of chronic rhinosinusitis. The main pathological changes of CRS are thickening of the sinus mucosa, collagen deposition, mucous gland hyperplasia and squamous metaplasia, excessive mucus secretion, and inflammatory cell infiltration. Due to the high incidence of CRS and the unclear pathogenesis, the establishment of a stable and reliable CRS animal model is an important guarantee for exploring its pathogenesis and developing effective therapeutic drugs.

[0003] At present, chronic sinusitis models have been successfully constructed in rodents such as New Zealand white rabbits and rats. However, the morphology of the nasal cavity and sinuses in the above-mentioned animal models is very different from that of humans. This structural difference may affect the accuracy of the disease model and the applicability of the research results. Therefore, in order to more accurately simulate human chronic sinusitis, it is extremely important to develop and use animal models that are closer to the anatomical structure of the human nasal cavity and sinuses. In this regard, primates, such as macaques and rhesus monkeys, are ideal choices because they have more similar physiological and anatomical characteristics to humans. The nasal cavity and sinus structures of primates are more similar to those of humans in size, shape and complexity, and can provide a research environment that is closer to the human pathological state.

[0004] In addition, the use of primate models can also explore immune responses and inflammatory pathways that are difficult to observe in mouse models. For example, the immune regulatory mechanisms in human CRS, such as the effects of inflammatory cells such as eosinophils and neutrophils on chronic sinusitis and the role of various other inflammatory mediators in chronic sinusitis, may be more accurately reproduced in primates. Therefore, the development of such a model can not only improve the accuracy of research, but also help develop and test new treatments, which can be initially verified and optimized in primate models before they are ultimately applied to human patients.

[0005] Therefore, providing an obstructive primate chronic sinusitis model with similar manifestations to human chronic sinusitis can better simulate the manifestations of human chronic sinusitis in terms of various biochemical indicators as well as the disease process and prognosis. A CRS primate model with high similarity to that of humans is of great significance for the efficacy and safety evaluation of CRS therapeutic drugs.

[0006] Compared to rodents like mice and rabbits, the nasal cavity of macaques is simpler in structure. The primary methods used to establish sinusitis models in rodents are nasal drops and nebulization. Rodents are less susceptible to obstruction than primates. While humans and macaques have similar nasal structures, there are also differences in their sinus structures. The drainage structure of macaque sinuses is simpler than that of humans, making them more difficult to obstruct. Due to the unique characteristics of the macaque nasal cavity, foreign matter is less likely to lodge, making obstruction models more difficult to establish.

[0007] An appropriately sized expandable sponge is placed in the middle nasal meatus, causing it to absorb water and swell, artificially simulating obstruction of the middle nasal meatus and poor drainage. Therefore, the present invention aims to develop a non-human primate chronic sinusitis model. This method, by placing an expandable sponge of a specific shape and size in one nasal cavity of a macaque, creates an obstructive primate chronic sinusitis model with pathological manifestations similar to those of human chronic sinusitis, and a primate CRS model with biochemical indicators, disease course, and outcomes that are highly similar to those of humans. Summary of the Invention

[0008] The present invention first provides a method for constructing a chronic sinusitis model in a non-human primate, which comprises the step of placing an expandable sponge in the middle nasal passage of the non-human primate.

[0009] In certain embodiments, the non-human primate comprises an Old World monkey.

[0010] In certain embodiments, the non-human primate comprises a macaque monkey.

[0011] In certain embodiments, the expansion sponge is placed in the right middle meatus of the non-human primate, with the left nasal cavity and paranasal sinus serving as a control side.

[0012] In some embodiments, the expanded sponge is in the shape of a cuboid.

[0013] In certain embodiments, the expanded sponge has a size of 5 mm x 2 mm x 1 mm.

[0014] In certain embodiments, the method further comprises the step of detecting modeling result indicators;

[0015] Optionally, the result indicators include one or more of purulent secretions in the middle nasal meatus under nasal endoscopy, soft tissue shadows in the maxillary sinus and sinus on sinus CT, and local tissue immunopathological staining.

[0016] The present invention also provides an application of the above-mentioned model construction method in the preparation or screening of drugs for treating chronic sinusitis.

[0017] The present invention also provides a non-human primate chronic sinusitis model, which is prepared by the above-mentioned model construction method.

[0018] Finally, the present invention provides an application of the above model in preparing or screening drugs for treating chronic sinusitis.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects:

[0020] (1) The method of the present invention does not require special instruments and equipment, and has low cost.

[0021] (2) The construction of the present invention successfully constructed a chronic sinus model in non-human primates for the first time, laying a foundation for exploring the occurrence and development of sinusitis, filling the technical gap in the existing sinusitis model construction, and has significant clinical and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Experimental design process;

[0023] Figure 2 Nasal endoscopic images of the modeling and control sides of macaques;

[0024] Figure 3 CT images of the sinuses on the modeling and control sides of macaques;

[0025] Figure 4 PCR amplification results of inflammatory factors in exfoliated cells mRNA from the macaque modeling side;

[0026] Figure 5 PCR amplification results of inflammatory factors from exfoliated cells mRNA on the control side of macaques;

[0027] Figure 6 PCR amplification results of inflammatory factors in the exfoliated cells mRNA at baseline on the modeling and control sides of macaques;

[0028] Figure 7 PCR amplification results of inflammatory factors in exfoliated cell mRNA from the macaque modeling and control sides one month ago;

[0029] Figure 8 PCR amplification results of inflammatory factors in exfoliated cell mRNA from the macaque modeling and control sides 3 months ago;

[0030] Figure 9 HE and Sirius red staining of the modeling and control sides of macaques. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0032] The experimental materials of the present invention are shown in Tables 1 to 3.

[0033] Table 1 Basic information of animals

[0034] Species and strains macaques Health status No nasal sinus disease, clean level quantity 6 weight 5.1-13.7kg age 9-12 years old

[0035] Table 2 Experimental equipment

[0036] Serial number Device Name brand 1 Xion Endoscopic Imaging System Germany's Xion company 2 WAROEX-55 Anesthesia System China Mindray 3 Dash4000 ECG Monitor GE Healthcare, USA 4 YHS-04 veterinary constant temperature lifting operating table

[0037] Table 3 Experimental materials

[0038]

[0039]

[0040] Environment: An animal room was provided for the macaques. The room temperature was controlled at 18-26°C and the humidity was controlled at 40-70%. Each macaque was housed in a stainless steel cage with a size of 90×90×90 cm. The animal room and cages were cleaned daily, the cages were replaced every two weeks, and the floor and walls of the animal room were disinfected every two weeks.

[0041] Example 1 Steps for Establishing a Chronic Sinusitis Model

[0042] (1) Six macaques aged 9-12 years and weighing 5.1-13.7 kg were randomly selected. All macaques were sourced from Beijing Xieerxin Biological Resources Research Institute Co., Ltd., and the procurement procedures were legal and compliant.

[0043] (2) The macaques in step (1) were fed at least twice daily: except for those requiring fasting during the experiment, the macaques were provided with free access to water daily. Each macaque consumed 200 g of feed, divided into two meals, at 7:00 a.m. and 5:00 p.m. In the afternoon, the macaques were fed with supplementary food and water. The supplementary food could be an appropriate amount of fresh vegetables or fruit. In addition, the amount of induction feed consumed by the macaques could be monitored regularly, such as by weighing the remaining amount of induction feed every 24 hours.

[0044] (3) Model selection criteria:

[0045] The macaques were deprived of food and water for 24 hours before anesthesia. After intramuscular injection of ketamine, a spiral CT scan of the macaques' sinuses was performed. Sinus CT scan: Scanning position: supine. Scanning range: from the top of the skull downward to the upper edge of the mandibular angle. No nasal or sinus inflammation was confirmed in the macaques.

[0046] The standard sinus CT data were exported in DICOM format and imported into ITK-SNAP 3D reconstruction software. The axial, sagittal, and coronal images of the sinus CT were observed in ITK-SNAP to ensure the integrity of the maxillary and ethmoid sinuses.

[0047] A nasal endoscope system was used to complete a nasal endoscopic examination of the macaque's nasal cavity, confirming that there was no pus or neoplasm in the middle nasal passage and olfactory fissure area of the macaque.

[0048] (4) Modeling operation using the packing method:

[0049] Propofol was used in combination with veterinary sevoflurane to complete tracheal intubation of macaques and achieve intravenous-inhalational anesthesia.

[0050] Exfoliated cells from the bilateral middle nasal passages were collected using exfoliated cell swabs.

[0051] The right middle meatus of the macaque's nasal cavity was packed with an expandable sponge, and the left sinus served as the control side.

[0052] Add 1 ml of Trizol to the collected exfoliated cell swabs for subsequent RNA extraction from nasal exfoliated cells and PCR amplification of inflammatory factors.

[0053] After the macaques regained consciousness from anesthesia, the endotracheal tube was removed and the monkeys were placed back in their cages.

[0054] (5) After feeding for 4 weeks according to step (2), the modeling result indicators were tested.

[0055] After intramuscular injection of ketamine anesthesia, spiral CT was used to perform sinus CT scans on the macaques to determine whether there was sinus inflammation on the modeling side.

[0056] Under intravenous and inhalational anesthesia, the macaques were examined again by nasal endoscopy to observe whether there was purulent secretion in the right middle nasal meatus and whether the model was successful.

[0057] Exfoliated cells from the bilateral middle nasal passages were collected using exfoliated cell swabs.

[0058] Add 1 ml of Trizol to the collected exfoliated cell swabs for subsequent RNA extraction from nasal exfoliated cells and PCR amplification of inflammatory factors.

[0059] Biopsies were taken from the right middle meatus mucosa of macaques.

[0060] After fixation with 10% neutral formalin, the samples were embedded in paraffin.

[0061] After tissue sectioning, the tissues were stained with HE and Sirius red.

[0062] The recruitment of inflammatory cells and the deposition of collagen in the tissues were analyzed and compared.

[0063] After the macaques regained consciousness from anesthesia, the endotracheal tube was removed and the monkeys were returned to their enclosure.

[0064] (6) After continuing feeding for 12 weeks according to step (2), the modeling result indicators were tested.

[0065] After intramuscular injection of ketamine anesthesia, spiral CT was used to perform sinus CT scans on the macaques to determine whether there was inflammation in the maxillary sinus of the macaques.

[0066] Under combined intravenous and inhalation anesthesia, the macaques were examined again by nasal endoscopy to observe whether there was purulent secretion in the right middle nasal meatus and whether the chronic sinusitis model was successfully established.

[0067] Exfoliated cells from the bilateral middle nasal passages were collected using exfoliated cell swabs.

[0068] Add 1 ml of Trizol to the collected exfoliated cell swabs for subsequent RNA extraction from nasal exfoliated cells and PCR amplification of inflammatory factors.

[0069] Under intravenous and inhalational anesthesia, biopsies were taken from the bilateral middle nasal passage mucosa of macaques. The right side was used to establish the chronic sinusitis model, and the left side was used as the control side.

[0070] After fixation with 10% neutral formalin, the samples were embedded in paraffin.

[0071] After tissue sectioning, the tissues were stained with H&E and Sirius red.

[0072] The recruitment of inflammatory cells and the deposition of collagen in the tissues were analyzed and compared.

[0073] Example 2 Evaluation indicators of chronic sinusitis model

[0074] (1) Nasal endoscopic evaluation indicators of chronic sinusitis

[0075] like Figure 2 Figures A and B show endoscopic images of the nasal cavity before modeling in macaques. No abnormal secretions or neoplasms were observed in the bilateral nasal cavities. The bilateral middle turbinate mucosa was smooth and drainage was unobstructed. Figure 2 As shown in C, the expanded sponge was trimmed to the appropriate size and then stuffed into the right middle nasal meatus of the macaque. The left side served as the control side. Figure 2 D. After 4 weeks, the macaques were examined by nasal endoscopy. Figure 2The right middle nasal meatus of the macaque shown in E is packed with an expansion sponge. A large amount of purulent secretions can be seen in the middle nasal meatus, indicating that the left middle nasal meatus is dry and clean. Figure 2 F. After 12 weeks, the macaques were examined by nasal endoscopy. Figure 2 The right middle nasal meatus of the macaque shown in G is packed with an expansion sponge. A large amount of purulent secretions can be seen in the middle nasal meatus, indicating that the left middle nasal meatus is dry and clean. Figure 2 H.

[0076] (2) CT evaluation indicators of chronic sinusitis

[0077] like Figure 3 As shown in Figures A, B, and C, the horizontal, sagittal, and coronal CT scans of the macaque's sinuses before modeling. No abnormal signal was observed in the macaque's bilateral ethmoid and maxillary sinuses. Four weeks later, the macaque underwent a sinus CT scan. Figure 3 As shown by the red arrows in D, E, and F, abnormal tissue signals can be seen in the maxillary sinus and ethmoid sinus of the modeling side of the macaque, indicating the presence of nasal sinusitis. On the control side, the maxillary sinus and middle nasal meatus are dry and clean. After 12 weeks, the macaques were examined by CT scan of the sinuses. Figure 3 As shown by the red arrows in H, I, and J, abnormal tissue signals can be seen in the maxillary sinus and ethmoid sinus of the modeling side of the macaque, indicating persistent sinusitis. In contrast, the maxillary sinus and middle meatus of the control side were dry and clean.

[0078] Example 3 Objective inspection and sample testing

[0079] (1) Exfoliated cells from the middle nasal passages on the modeling side were collected at baseline and at weeks 4 and 12. After extracting mRNA from the nasal exfoliated cells, qRT-PCR was used to amplify and detect the expression of inflammatory factors in the mRNA of the nasal exfoliated cells. Figure 4 The continuous changes of inflammatory factor expression in the mRNA of exfoliated cells of the nasal cavity on the modeling side, such as Figure 4 As shown, the expression levels of CXCL1, CXCL2, and CXCL8 increased continuously, reaching a peak at week 12, significantly higher than baseline levels. Similarly, type 1 inflammatory factors and cytokines IL-1β, TNF-α, and TGF-β also showed sustained high-level expression. Factors associated with type 2 inflammation, such as IL-25, IL-33, TSLP, CST1, and Periostin, also showed high expression. This suggests that obstructive inflammation causes the upregulation of multiple types of cytokines.

[0080] (2) Exfoliated cells from the middle nasal passage of the control side were collected at baseline and at weeks 4 and 12. After extracting mRNA from the nasal exfoliated cells, the expression of inflammatory factors in the mRNA of the nasal exfoliated cells was amplified and detected using qRT-PCR. Figure 5The continuous changes in the expression of inflammatory factors in the mRNA of exfoliated cells in the nasal cavity on the control side showed that the expression levels of factors related to type 2 inflammation, such as IL-25, IL-33, TSLP, and Periostin, were significantly increased, while the expression levels of other types of inflammatory factors were not significantly increased, suggesting that obstructive inflammation caused the upregulation of type 2 inflammatory factors on the contralateral side.

[0081] (3) At baseline, week 4, and week 12, exfoliated cells from the bilateral middle nasal passages were collected, and after extracting the mRNA from the nasal exfoliated cells, the expression of inflammatory factors in the mRNA from the bilateral nasal exfoliated cells was amplified and detected using qRT-PCR. The expression differences of inflammatory factors between the modeling side and the control side were compared at the same time. The results before modeling are as follows Figure 6 As shown, in Figure 6 In the 4th week of modeling, it can be seen that the expression of inflammatory factors on both sides is similar. Only the expression of IL-1β on the control side is slightly higher than that on the modeling side. Figure 7 In the experiment, the expression of inflammatory factors on both sides was similar. Only the expression of IL-1β on the modeling side was higher than that on the control side ( Figure 7 ); In the 12th week of modeling, it can be seen that except for Periostin ( Figure 8 ), the inflammatory factors on the model side were significantly higher than those on the control side ( Figure 8 ).

[0082] (4) Nasal mucosal tissues from the middle nasal passages were collected for pathological examination at baseline and at weeks 4 and 12. The nasal mucosal tissues were stained with hematoxylin-eosin and picrosirius red to observe the integrity of the nasal mucosal epithelium, the degree of inflammatory cell infiltration, and collagen deposition. Figure 9 A and B are HE staining of the middle nasal passage mucosa on the control side, showing intact epithelium (blue arrow) and a small amount of inflammatory cell infiltration (red arrow). Figure 9 E and F show that the integrity of the middle nasal passage epithelium was significantly damaged (blue arrows), and at the same time, the interstitial edema was obvious, accompanied by the infiltration of a large number of inflammatory cells (red arrows). Figure 9 I and J show that the integrity of the middle nasal meatus epithelium was severely damaged on the modeling side, the infiltration of inflammatory cells was reduced, and the collagen deposition was significantly increased ( Figure 9 K, L).

[0083] Statistical methods:

[0084] IBM SPSS Statistics 26 (IBM Corp.) and Prism 9 (GraphPad Software, Inc.) were used for data analysis. Paired t-tests were used to compare changes in mRNA expression of inflammatory factors in nasal exfoliated cells from the modeling and control sides of the same experimental animals. P values < 0.05 indicated statistical significance.

[0085] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for establishing a model of chronic sinusitis in non-human primates, characterized in that: The method comprises the steps of placing an expandable sponge in the middle nasal meatus of the non-human primate; The chronic sinusitis may include multiple pathological types.

2. The model building method according to claim 1, characterized in that: The non-human primates include Old World monkeys.

3. The model building method according to claim 1, characterized in that The non-human primates include macaques.

4. The model building method according to claim 1, wherein: The expansion sponge was placed in the right middle meatus of the non-human primate, and the left nasal cavity and paranasal sinus served as a control side.

5. The model building method according to claim 1, characterized in that: The shape of the expansion sponge is a cuboid.

6. The model building method according to claim 5, characterized in that: The size of the expansion sponge is 5 mm×2 mm×1 mm.

7. The model building method according to claim 1, characterized in that: The method further comprises the step of detecting modeling result indicators; The outcome indicators include one or more of purulent secretions in the middle nasal meatus under nasal endoscopy, soft tissue shadows in the maxillary sinus and sinus on sinus CT, and local tissue immunopathological staining.

8. Use of the model establishment method according to any one of claims 1 to 7 in the preparation or screening of drugs for treating chronic sinusitis.

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