Method for constructing non-human primate chronic rhinosinusitis model

Abstract

The present invention relates to the technical field of disease model construction, in particular to a method for constructing a non-human primate chronic rhinosinusitis model. The method comprises the step of placing an expansive sponge in the nasal meatus of the non-human primate. The method does not require any special instrument device, and has low costs. By means of the construction of the present invention, a chronic rhinosinusitis model is successfully constructed in a non-human primate for the first time, which lays the foundation for exploring the occurrence and development of rhinosinusitis, fills the technical gap in existing rhinosinusitis model construction, and has great clinical value and economic value.

Description

A method for establishing a chronic sinusitis model in non-human primates 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, hyperplasia of mucous glands 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 model side;

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

[0027] Figure 6 PCR amplification results of inflammatory factors in the exfoliated cell 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 model side and the control side one month ago;

[0029] Figure 8 PCR amplification results of inflammatory factors in exfoliated cell mRNA from the macaque model side and the control side 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] Table 2 Experimental equipment

[0035] Table 3 Experimental materials

[0036] 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.

[0037] Example 1 Steps for Establishing a Chronic Sinusitis Model

[0038] (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.

[0039] (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.

[0040] (3) Model selection criteria:

[0041] 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.

[0042] 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.

[0043] 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.

[0044] (4) Modeling operation using the packing method:

[0045] Propofol was used in combination with veterinary sevoflurane to complete tracheal intubation of macaques and achieve intravenous-inhalational anesthesia.

[0046] Exfoliated cells from the bilateral middle nasal passages were collected using exfoliated cell swabs.

[0047] 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.

[0048] 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.

[0049] After the macaques regained consciousness from anesthesia, the endotracheal tube was removed and the monkeys were placed back in their cages.

[0050] (5) After feeding for 4 weeks according to step (2), the modeling result indicators were tested.

[0051] 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.

[0052] 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.

[0053] Exfoliated cells from the bilateral middle nasal passages were collected using exfoliated cell swabs.

[0054] 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.

[0055] Biopsies were taken from the right middle meatus mucosa of macaques.

[0056] After fixation with 10% neutral formalin, the samples were embedded in paraffin.

[0057] After tissue sectioning, the tissues were stained with HE and Sirius red.

[0058] The recruitment of inflammatory cells and the deposition of collagen in the tissues were analyzed and compared.

[0059] After the macaques regained consciousness from anesthesia, the endotracheal tube was removed and the monkeys were returned to their enclosure.

[0060] (6) After continuing feeding for 12 weeks according to step (2), the modeling result indicators were tested.

[0061] 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 macaques' maxillary sinus.

[0062] Under combined intravenous and inhalation anesthesia, the macaques were subjected to nasal endoscopy again to observe whether there was purulent secretion in the right middle nasal meatus and whether the chronic sinusitis model was successful.

[0063] Exfoliated cells from the bilateral middle nasal passages were collected using exfoliated cell swabs.

[0064] 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.

[0065] 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.

[0066] After fixation with 10% neutral formalin, the samples were embedded in paraffin.

[0067] After tissue sectioning, the tissues were stained with H&E and Sirius red.

[0068] The recruitment of inflammatory cells and the deposition of collagen in the tissues were analyzed and compared.

[0069] Example 2 Evaluation indicators of chronic sinusitis model

[0070] (1) Nasal endoscopic evaluation indicators of chronic sinusitis

[0071] As shown in Figure 2, Figures A and B are nasal endoscopic images of the nasal cavity of the macaque before modeling. No abnormal secretions or neoplasms were found in the bilateral nasal cavities. The bilateral middle turbinate mucosa was smooth and the drainage was unobstructed. As shown in Figure 2C, the expansion sponge was trimmed to an appropriate size and then stuffed into the right middle nasal meatus of the macaque. The left side served as the control side, Figure 2D. Four weeks later, the macaque underwent nasal endoscopy. As shown in Figure 2E, the right middle nasal meatus of the macaque was stuffed with an expansion sponge. A large amount of purulent secretions were visible in the middle nasal meatus, indicating that the left middle nasal meatus was dry and clean, Figure 2F. Twelve weeks later, the macaque underwent nasal endoscopy. As shown in Figure 2G, the right middle nasal meatus of the macaque was stuffed with an expansion sponge. A large amount of purulent secretions were visible in the middle nasal meatus, indicating that the left middle nasal meatus was dry and clean, Figure 2H.

[0072] (2) CT evaluation indicators of chronic sinusitis

[0073] As shown in Figure 3, Figures A, B, and C are the horizontal, sagittal, and coronal CT scans of the macaque's sinuses before modeling. No abnormal signal shadows were seen in the ethmoid and maxillary sinuses on both sides of the macaque. Four weeks later, the macaque underwent a sinus CT scan. As shown by the red arrows in Figures 3D, E, and F, abnormal tissue signal shadows were visible in the maxillary sinus and ethmoid sinus on the modeling side of the macaque, indicating the presence of sinusitis. On the other hand, the maxillary sinus and middle meatus were dry and clean on the control side. Twelve weeks later, the macaque underwent a sinus CT scan. As shown by the red arrows in Figures 3H, I, and J, abnormal tissue signal shadows were visible in the maxillary sinus and ethmoid sinus on the modeling side of the macaque, indicating the persistence of sinusitis. On the other hand, the maxillary sinus and middle meatus were dry and clean on the control side.

[0074] Example 3 Objective inspection and sample testing

[0075] (1) At baseline, week 4, and week 12, exfoliated cells were collected from the middle nasal passages on the modeling side. After extracting the mRNA of 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 4 shows the continuous changes in the expression of inflammatory factors in the mRNA of the nasal exfoliated cells on the modeling side. As shown in Figures AC, the expression levels of CXCL1, CXCL2, and CXCL8 continued to increase, and the expression levels of inflammatory factors reached a peak at week 12, which was significantly higher than the baseline level. Similarly, type 1 inflammatory factors and cytokines IL-1β, TNF-α, and TGF-β also showed changes in continuous high-level expression (Figures 4D, H, and J). The expression of factors related to type 2 inflammation, IL-25, IL-33, TSLP, CST1, and Periostin, also showed changes in high expression (Figures 4F, G, I, K, and L). This suggests that obstructive inflammation causes the upregulation of multiple types of cytokines.

[0076] (2) At baseline, week 4, and week 12, exfoliated cells were collected from the middle nasal passages of the control side. After extracting the mRNA of 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 5 shows the continuous changes in the expression of inflammatory factors in the mRNA of the nasal exfoliated cells of the control side. As shown in Figures F, G, I, and L, the expression levels of IL-25, IL-33, TSLP, and Periostin, factors related to type 2 inflammation, were significantly increased, while the expression levels of other types of inflammatory factors were not significantly high. This suggests that obstructive inflammation caused the upregulation of type 2 inflammatory factors on the contralateral side.

[0077] (3) At baseline and at weeks 4 and 12, exfoliated cells from the bilateral middle nasal passages were collected. 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 bilateral nasal exfoliated cells. The expression differences of inflammatory factors on the modeling side and the control side were compared at the same time. The results before modeling are shown in Figure 6. In Figures 6A-C and EL, the expression levels of inflammatory factors on both sides are similar. Only the expression of IL-1β on the control side is slightly higher than that on the modeling side (Figure 6D). At week 4 of modeling, it can be seen in Figures 7A-C and EL that the expression levels of inflammatory factors on both sides are similar. Only the expression of IL-1β on the modeling side is higher than that on the control side (Figure 7D); at week 12 of modeling, it can be seen that, except for Periostin (Figure 8L), the inflammatory factors on the modeling side are significantly more highly expressed than those on the control side (Figure 8A-K).

[0078] (4) The nasal mucosa tissues of the middle nasal passage were collected for pathological examination at baseline and at weeks 4 and 12. The nasal mucosa tissues were stained with hematoxylin-eosin and Sirius red to observe the integrity of the nasal mucosal epithelium, the degree of infiltration of inflammatory cells, and the deposition of collagen. Figures 9A and B are HE staining of the middle nasal passage mucosa on the control side, which has intact epithelium (blue arrow) and a small amount of inflammatory cell infiltration (red arrow). At week 4 of modeling, as shown in Figures 9E and F, the integrity of the epithelium of the middle nasal passage mucosa on the modeling side was significantly damaged (blue arrow). At the same time, there was obvious edema in the interstitial space, accompanied by the infiltration of a large number of inflammatory cells (red arrow). At week 12 of modeling, as shown in Figures 9I and J, the integrity of the epithelium of the middle nasal passage mucosa on the modeling side was severely damaged, the infiltration of inflammatory cells was reduced, and the collagen deposition was significantly increased (Figures 9K and L).

[0079] Statistical methods:

[0080] 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.

[0081] 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; Optionally, 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, characterized in that: The expansion sponge was placed in the right middle nasal meatus of the non-human primate, and the left nasal cavity and paranasal sinus served as the control side.

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

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

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

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

9. A non-human primate chronic sinusitis model, characterized in that: The model is prepared by the model building method described in any one of claims 1-8.

10. Use of the model according to claim 9 in preparing or screening drugs for treating chronic sinusitis.

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