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Introduction
The vomeronasal organ (VNO), a pheromone-sensing organ in the nasal cavity, connects to the accessory olfactory bulb at the dorso-caudal part of the olfactory bulb and plays an important role in chemical communication, including social interactions and sexual behavior, through the detection of pheromones [1]. Vomeronasalitis, or inflammation of the VNO, is associated with behavioral changes due to cytoarchitectural alterations, including a decrease in VNO epithelium thickness and a reduction in sensory neurons [2].
Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS), a neurodegenerative disease characterized by central nervous system (CNS) inflammation [3]. MS patients show neurological disorders, including blindness, diplopia, and dizziness [4]. An olfactory deficit is the representative neurological disorder in both MS patients [5] and EAE-induced animals [6, 7]. Although studies of EAE have been conducted for the main olfactory system [6, 8, 9] and in the CNS tissues [3], little is known about the accessory olfactory system, which is involved in chemoreception [10]. Therefore, we evaluated whether an inflammatory response occurs in the VNO during EAE.
Materials and Methods
Animals and EAE induction
Experimental animals (female and male C57BL/6J mice aged 7 weeks; Central Laboratory Animal Inc.) were housed for 1 week in the animal facility of Jeju National University under controlled conditions (12 hour light/dark cycle, temperature=24°C±2°C) before immunization. This study was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Jeju National University (Permission no. 2022-0062).
EAE induction was achieved as described previously [6]. Briefly, a mixed solution containing 1 mg/ml myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide (GL Biochem Ltd.), Freund’s complete adjuvant (Difco Laboratories Inc.), 5 mg/ml Mycobacterium tuberculosis H37Ra (Difco Laboratories Inc.), and 500 ng pertussis toxin (List Biological Laboratories Inc.) was administrated via intraperitoneal injection on the immunization day (day 0) and 2 days post-immunization.
Tissue preparation
At the peak paralytic stage, EAE-induced mice and normal mice (n=8 per group, 4 male and 4 female of each group) were sacrificed under deep anesthesia using CO2 gas. Experimental animals were fixed with 4% paraformaldehyde in phosphate buffer solution (PBS; 0.1 M, pH 7.4). Skulls with the VNO were decalcified as described previously using formic acid-sodium citrate buffer [6]. Samples, including the VNO after decalcification, were embedded in tissue infiltration medium paraffin wax (Leica) and sections 5 µm thick were produced using a Leica RM2135 microtome. Paraffin sections were deparaffinized in xylene, dehydrated by passage through a gradient ethanol series. The sections were subjected to hematoxylin-eosin and periodic acid-Schiff staining or immunohistochemistry.
Immunohistochemistry
A Vectastain Elite ABC Kit (Vector Laboratories) was adapted for immunohistochemical staining as described previously [6]. Briefly, the deparaffinized sections were reacted with 0.01 M citrate buffer (pH 6.0) in a microwave oven for 3 minutes and then exposed to methanol with 0.03% hydrogen peroxide for 20 minutes. Next, 10% normal goat serum in PBS (blocking solution; Vector Laboratories) was added. Subsequently, the sections were incubated with primary antibodies, including rabbit anti-olfactory marker protein (OMP; ab183947; Abcam), anti-myeloperoxidase (MPO; ab9535; Abcam), and anti-ionized calcium binding adaptor molecule 1 (Iba1; 019-19741; FUJIFILM Wako Pure Chemical Corp.), for 1 hour at room temperature. After washing three times with PBS, the sections were reacted with biotinylated rabbit immunoglobulin G and avidin-biotin complex peroxidase. A DAB Kit (Vector Laboratories) was used for the peroxidase reaction, and the sections were counterstained for 10 seconds with hematoxylin. To calculate the Iba1 and OMP-positive area, three different sections were used per animal, and the immunoreaction-positive area ([positive area/total area]×100 [%]) was calculated. The number of MPO-positive cells, indicating neutrophils, in the VNO was counted as follows, using at least three different stained sections per mouse VNO, and at least four mice per group.
Statistical analysis
All statistical analysis was performed by GraphPad (v.9.3.1; GraphPad Software). Our semi-quantitative data, which are presented as the mean±standard error of the mean, were analyzed using a one-way analysis of variance followed by Tukey’s test for multiple comparisons at a significance level of P<0.05.
Results
In histopathological examination, inflammatory cells were detected in the spinal cord of EAE-induced mice (Fig. 1D) compared to normal control tissues (Fig. 1A). Perivascular cuffing (Fig. 1D, arrowhead) and inflammatory cell infiltration (Fig. 1D, arrows) are representative histopathological changes in the spinal cord of EAE-induced mice. The VNO, which consists of two types of epitheliums (the vomeronasal sensory epithelium [VSE] and vomeronasal non-sensory epithelium [VNSE]), was capsulized with cartilage in both normal control and EAE-induced mice (Fig. 1B, E). Greater magnification revealed inflammatory cells (Fig. 1F, arrows) in the VSE and VNSE of the latter (Fig. 1F), compared to that of normal control (Fig. 1C). Inflammatory cells were located in the connective tissues and lamina propria of the VNO of EAE-induced mice. Furthermore, mucus increased in the epithelium of EAE-induced mice (Fig. 1E, F).
To investigate these inflammatory cells, we performed immunohistochemical staining using Iba1- and MPO-specific antibodies as markers for macrophages and neutrophils, respectively. Few Iba1-positive cells (Fig. 2B, arrowhead) were detected in the VNO of normal controls (Fig. 2A, B), whereas many Iba1-positive cells (Fig 2F, arrowheads) were detected in the VNO of the EAE-induced mice (Fig. 2E, F). Our semiquantitative results confirmed a significant increase in Iba1-positive cells in the VNO of EAE-induced mice (Fig. 2I). In the lower magnification view, MPO-positive cells were hard to find in both normal control and EAE-induced mice (Fig. 2C, G). MPO-positive neutrophils were detected in the VNO of the EAE-induced mice (Fig. 2H, arrows), but they were rare in controls (Fig. 2D). Quantitative results confirmed a significant increase in MPO-positive cells in the VNO of EAE-induced mice (Fig. 2J).
The intensity of the OMP-positive immunoreaction was lower in the VSE of EAE-induced mice (Fig. 3B) than in controls (Fig. 3A, C). The vomeronasal glands were located in the VNSE of both groups (Fig. 3D, E). However, increased mucus secretion (Fig. 3E, arrow) and glycogen accumulation (Fig. 3E, arrowheads) were confirmed in the EAE-induced mice.
Discussion
As in the olfactory mucosa [6], we found that the VNO is inflamed during the course of EAE. This is in accordance with a report showing an increased level of pro-inflammatory mediators in serum, suggesting that inflammation occurs in a variety of tissues, including the lungs [11]. If systemic inflammation occurs in EAE, the VNO should be affected by blood-borne pro-inflammatory mediators [12] resulting in the migration of inflammatory cells into the VNO (e.g., the MPO-positive neutrophils and Iba1-positive macrophages shown in this study). It is likely that an increased number of inflammatory cells in the connective tissues of the VNO during EAE, through the secretion of extracellular matrix, facilitates tissue destruction leading to the dysfunction of the organ.
As far as the neuronal activity of receptor cells in the VNO, it is postulated that the downregulation of OMP is associated with olfactory sensory neuron dysfunction [2] given that OMP is a marker for mature olfactory sensory neurons and vomeronasal sensory neuron [13]. Vomeronasal sensory neurons are deeply associated with the social communication by detection of pheromone [1]. Pigs with vomeronasalitis showed the intraspecific aggression by alteration of VSE [2]. In the present study, we found that the decreased intensity of OMP immunoreactivity in the VSE with EAE was associated with reduced activity of VSE receptor cells. Dysfunction of the accessory olfactory system may be attributable to inflammation in the VNO. The signal changes in the VSE should be studied further. Collectively, in MOG-induced EAE, vomeronasalitis occurs in the VNO leading to problems with olfaction and social communication.
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