Allergic rhinitis (AR) is a type 2 T helper cell (Th2)-skewed disease accompanied by eosinophils infiltration into nasal tissue, following exposure to inhaled antigens in the upper airway in genetically predisposed individuals. The prevalence of AR has been increasing, and over 500 million people suffer from AR worldwide [1
]. Allergic rhinitis is diagnosed through a typical history of three characteristic symptoms (sneezing and nasal scratching, water rhinorrhea, or nasal obstruction) and clinical examination findings: detection of eosinophils in nasal discharge, nasal provocation test using antigen, elevation of allergen-specific immunoglobulin E (IgE) antibodies, or skin prick test [1
]. Likewise, asthma is diagnosed on the basis of clinical symptoms (wheezing and dyspnea) and clinical examination findings: index of airway responsiveness in the respiratory tract to detect airway hyperresponsiveness (AHR), bronchodilator test, exhaled nitric oxide concentrations, and/or increased serum levels of specific IgE [3
AHR is an exaggerated response to a variety of irritants including histamine, methacholine, bradykinin, isotonic or hypertonic solutions, and cold or dry air; it is one of the hallmark features of allergic airway inflammation [4
]. The mechanisms of AHR are implicated in multiple changes in the airway wall, characterized by epithelial cell layer disruption, increased collagen deposition, hypertrophy of smooth muscle, and increased vascularization [7
]. Sensory nerves and endings of the parasympathetic nervous system become exposed; this leads to increased sensitivity to endogenous or exogenous stimuli, causing persistence of airway inflammation [8
]. Clinically, lower AHR (L
AHR) in asthma is reflected by an increased airway resistance due to constriction of the bronchial smooth muscles following nonspecific stimulants such as methacholine (MCh) or histamine. Clinical examination for L
AHR through nonspecific stimulant provocation test is routinely performed to diagnose and assess the severity of asthma in clinical practice. Conversely, upper AHR (U
AHR) in AR is usually evaluated on the basis of onset of clinical symptoms (nasal obstruction, nasal secretion and sneezing) after provocation. However, measurement of U
AHR in AR by which indicated as nasal resistance following provocation is rarely performed in clinical practice, although clinical trials using nasal peak flow or rhinomanometry have been performed [1
In animals, to detect AHR following MCh administration in the animal model, measurement of L
AHR, reflected by lung resistance and compliance, is commonly performed using non-invasive or invasive plethysmography [11
]. Animals are evaluated 24 to 48 h after the last allergen challenge because the difference between naïve and asthma groups in L
AHR the highest during late-phase inflammation following the last antigen challenge [13
]. In contrast, no studies have reported the evaluation of U
AHR following MCh administration in the upper airway eosinophilic inflammation mimicking AR, while evaluation in the early phase by specific antigen has been performed by using frequency of sneezing and scratching. Although mechanism of U
AHR is unclear, threshold elevation by MCh in AR model indicates hyperresponsiveness. Despite the demand for a U
AHR evaluation system that can directly measure swelling of the nasal mucosa rather than nasal resistance, no measurement system has been developed.
Recently, micro-computed tomography (micro-CT) has been widely used for analysis of bone and soft tissue in animals [15
]; it can provide high spatial and temporal resolution images [16
]. Analysis using micro-CT is an innovative method for evaluation of anatomical structures and pathological lesions compared with histological analysis [17
]. Here we investigated the application of micro-CT for U
AHR evaluation to measure swelling of the nasal mucosa following MCh intranasal administration in an AR mouse model and assessment of anti-inflammation compounds.
BALB/c mice (weight 20 g, selected from female 6–8 weeks), purchased from Shimizu Experimental Material (Kyoto, Japan), were bred in a specific pathogen-free animal facility with a regular 12 h light/dark cycle. All experiments were performed in accordance with the Animal Care and Use Committee of Kansai Medical University (18-082).
2.2. Ovalbumin (OVA)-Induced AR Model
Protocol of schema is shown in Figure S1A
. Briefly, mice were sensitized through intraperitoneal injection of 50 μg OVA (Sigma-Aldrich, Missouri, USA.) or phosphate buffered saline (PBS) with 1 mg of aluminum hydroxide (Thermo Fisher Scientific, Massachusetts, USA) on Days 0 and 14 and daily challenged with OVA (nebulization of 1% OVA for 30 min) during Days 21–25. On Day 26, micro-CT analysis was performed 24 h after the last challenge. Then, OVA-induced AR mouse model was prepared (see histology in Figure S1B
). In some experiments, mice were pretreated with an intraperitoneal injection of dexamethasone (10 mg/kg, Sigma-Aldrich) or sesame oil with 4% dimethyl sulfoxide as vehicle control 1 h before each OVA challenge on Days 21, 23 and 25.
2.3. Evaluation of Nasal Mucosa by Micro-Computed Tomography
Mice were treated with 2 μL of PBS or 0.5–2.0 mg/mL MCh (Sigma-Aldrich) that was administered into each nostril for 15 min under systemic anesthesia with a mixture of 50 mg/kg ketamine (Ketalar, Daiichi-Sankyo, Tokyo, Japan) and 0.5 mg/kg medetomidine (Domitor; Pfizer, New York City, USA). In the study of time course, mice were treated with 2μl MCh (1mg/mL) or PBS intranasal administration for 0, 15, 60 or 120 min under anesthesia. Then, mice were sacrificed with an intraperitoneal injection of pentobarbital (Kyoritsu Pharmaceutical, Nara, Japan), and micro-CT imaging was immediately performed using the specifically on single photon emission computed tomography/computed tomography (SPECT/CT) system (Siemens, Bayern, Germany) with the following scanner settings: tube voltage of 70 kVp and current of 500 μA over 360 continuous projections with an exposure time of 1000 ms per projection. An aluminum filter (0.5 mm) was used to reduce beam-hardening artifacts. The cross-sectional images were reconstructed using Inveon Viewer QuickLaunch software (Siemens Healthcare, Erlangen, Germany) and converted to the Digital Imaging and Communications in Medicine format using PMOD software version 3.703 (PMOD Technologies, Zurich, Switzerland). For each mouse, a stack of 768 cross-sections was reconstructed, and manual regions of interest (ROIs) were evaluated using OsiriX software version 8.0.1 (Pixmeo SARL, Bernex, Switzerland). Data were reconstructed using Inveon Viewer QuickLaunch software version 126.96.36.199. The reconstructed two-dimensional (2D) images (1088 × 1088 pixels) comprised 19.71 μm voxels with 10,000 Hounsfield Units (HU) (window width) and 2000 HU (window level).
2.4. Statistical Analysis
Data are presented as means ± standard errors of mean (SEMs). Statistical significance was determined using Mann–Whitney U test by software GraphPad Prism version 7 (GraphPad, San Diego, USA). The threshold of significance was set at p < 0.05 for all tests.
A supplementary methods section can be found in this article’s online repository.
We found that the structures of the nasal cavity and the paranasal sinuses could be clearly identified on 2D reconstructed images using an image analyzer for animals and a micro-CT scanner. Although studies have reported the use of micro-CT to determine the anatomy of the nasal cavity and paranasal sinuses [18
], there have been no reports of a quantitative approach to measure the degree of swelling of the nasal mucosa. In this study, we initially developed a procedure involving the measurement area and condition, followed by the development of a quantitative method focused on measuring swelling of the nasal mucosa using mucosa index. Regarding U
AHR evaluation, we showed an increase in U
AHR in an AR mouse model due to MCh-induced swelling of the nasal mucosa using a micro-CT scanner and suggested an original method for U
A functional in vivo assay for an AR mouse model was set up by measuring the frequency of sneezing and nose-scratching for 10 min after the last challenge with specific antigen or nonspecific antigen [20
]. However, this assay can only detect early-phase response, not late-phase responses and nasal resistance, which is one of the biological responses following AHR. Increasing L
AHR in asthma patients reflects tissue remodeling with destruction of the epithelial layer, especially in those with persistent inflammation [7
]. In an AR patient, U
AHR is also enhanced by repeated nasal challenges with specific antigens [6
] and is involved in persistence of inflammation. Similar results have been reported in an AR animal model [23
]. However, airway resistance in asthma and AR results from the contraction of the bronchial smooth muscles and swelling of the nasal mucosa with nasal discharge, respectively [24
]. Unlike the evaluation of airway resistance in asthma, evaluation of nasal resistance is complicated because it requires consideration of not only this different pathway but also the influence of the lower airway. Therefore, few studies have focused on evaluation using plethysmography in an AR model, whereas this approach has been widely used to determine the underlying mechanism or to evaluate the effect of new therapeutic compounds in an antigen-induced asthma model [26
Regarding previous studies using airway functional tests indicated for nasal airway resistance or respiratory frequency accompanied with resistance, Miyahara et al., using non-invasive measures such as whole-body plethysmography or invasive plethysmography, reported that both allergen-specific and nonspecific provocations were involved in U
AHR of early- and late-phase nasal responses [23
]. Likewise, Mizutani et al. reported nasal hyperresponsiveness to histamine, but not to MCh, induced by repetitive inhalations of cedar pollen into the nose of guinea-pigs [28
]. However, it is impossible to block the background factors such as the effect of the lower airway, nasal discharge, and body motion of mouse, and induction of AR in non-asthmatic animals has not been achieved because of technical challenges and interactions between upper and lower airway diseases through drainage of mediators, neural reflex via nervous vagus, and/or systemic dissemination of mediators [29
]. The evaluation of U
AHR using invasive plethysmography for upper airway is affected by surgical stress and anesthesia but not by the lower airway [30
]. Taken together, currently, measurement of nasal resistance using whole-body plethysmography or invasive plethysmography does not seem to be an appropriate approach to evaluate U
In contrast, using the approach involving measurement of swelling of the nasal mucosa, we could detect an U
AHR-induced direct effect (see schema in Figure S4
). This system could assess reactivity of MCh 24 h after the last OVA challenge, as reflected in U
AHR in the late-phase nasal responses. Notably, we could not find any increase in the mucosa index by OVA treatment 24 h following repeated OVA challenges, suggesting that the increased nasal resistance and respiratory frequency reported in previous studies might reflect nasal discharge [23
]. Moreover, we found abrogation of U
AHR with steroids, which indicated that swelling of the nasal mucosa was induced by U
AHR through epithelial layer destruction. However, innovation of an apparatus and cost reduction is required because micro-CT requires expensive equipment, and a long time (almost 20 min/mouse) is required to capture high-resolution images.
In conclusion, our novel approach of evaluation of UAHR involving measurement of swelling of the nasal mucosa after MCh pretreatment using micro-CT not only sheds light on the potential mechanisms underlying AR but will also help with the development of a new therapeutic drug in an AR mouse model.