Tumor necrosis factor alpha inhibits olfactory regeneration in a transgenic model of chronic rhinosinusitis-associated olfactory loss

Abstract

Background: Olfactory loss is a debilitating symptom of chronic rhinosinusitis (CRS). Although olfactory sensory neurons (OSNs) are normally regenerated constantly in the olfactory epithelium (OE), a transgenic model of CRS-associated olfactory loss (inducible olfactory inflammation [IOI] mouse) shows that inflammation causes widespread OSN loss without progenitor cell proliferation. In this study, we further examine whether the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) inhibits olfactory regeneration.

Methods: IOI mice underwent either unilateral bulbectomy or sham surgery and then were induced to express TNF-alpha in the OE for 1 week. After death, the mice were assessed histologically and with bromodeoxyuridine staining to determine the effect of TNF-alpha on olfactory regeneration.

Results: In the absence of TNF-alpha, bulbectomy was associated with death of OSNs, followed by robust proliferation of neural progenitors and regrowth of the OE. At 12 days postbulbectomy, OE thickness on the operated side had recovered to >80% of the unoperated side. In mice in which TNF-alpha expression was induced, significantly reduced proliferation was observed, associated with failure of normal reconstitution of OE thickness.

Conclusion: The mechanism of olfactory dysfunction in CRS remains incompletely understood. Previous studies with a transgenic mouse model suggested that inflammation inhibits progenitor cell proliferation and olfactory regeneration. Here, the role of the CRS-associated cytokine TNF-alpha was investigated using surgical ablation of the olfactory bulb to stimulate synchronous OSN turnover. We find that TNF-alpha expression prevents normal OE recovery, supporting the role of suppressed olfactory regeneration in the pathophysiology of CRS-associated olfactory loss.

Figure 1

Olfactory bulbectomy is a model for olfactory neuronal regeneration. Wild-type and inducible olfactory inflammation (IOI) mice were bulbectomized and killed, and their nasal cavities were then embedded in paraffin and processed as described in the Methods section. Shown is a cross-section of a wild-type mouse nasal cavity at the level of the olfactory bulb, processed 7 days after bulbectomy. Identified is the normal left olfactory bulb (B), right olfactory bulb after bulbectomy (BX), nasal septum (NS), and turbinates (T).

Figure 2

Tumor necrosis factor (TNF) α inhibits repopulation of olfactory neurons after bulbectomy in the inducible olfactory inflammation (IOI) mouse. Wild-type and IOI mice were bulbectomized (OBX) or underwent Sham surgery (Sham) as described in the Methods section. Shown are images of the nasal septum at (A–D) 10× and (E–H) 20× magnification with the unoperated side on the left and the operated side on the right. (A and E) Wild-type, sham-operated mouse with multiple layers of olfactory neurons and an overlying layer of sustentacular cells on both sides of the nasal cavity. (B and F) Wild-type, bulbectomized mouse; the olfactory epithelium has largely recovered and is similar in appearance to the contralateral side. (C and G) Sham-operated IOI mouse olfactory epithelium is histologically identical to that of wild-type mouse. (D and H) Bulbectomized IOI mouse with substantially thinned olfactory epithelium and disorganized axon bundles. Data are representative of at least three different mice for each condition. Scale bars: panels A–D, 100 μm; panels E–H, 50 μm.

Figure 3

Tumor necrosis factor (TNF) α inhibits repopulation of the olfactory epithelium after bulbectomy in inducible olfactory inflammation (IOI) mice. Thickness of the olfactory epithelium was measured on each side of the nasal septum for both sham-operated and bulbectomized mice. Data are shown as the mean thickness ± SE for at least three different mice. *p < 0.05 versus unoperated side; **p < 0.001 versus unoperated side; #p < 0.005 versus wild type.

Figure 4

Tumor necrosis (TNF) α inhibits mitosis of basal progenitor cells in the olfactory epithelium of inducible olfactory inflammation (IOI) mice. Wild-type and IOI mice were processed for BrdU staining as described in the Methods section. Representative images are shown for wild-type (WT) and IOI mice that underwent either (A and C) sham surgery or (B and D) bulbectomy. Proliferating basal progenitor cells (solid arrows) and infiltrating inflammatory cells (dashed arrows) are noted. (E). BrdU⁺ cells were counted along the entire length of the nasal septum on both the operated and unoperated side. Data are represented as the mean number of BrdU cells ± SE for at least three different mice. *p < 0.05 versus wild type; **p < 0.01 versus unoperated side. Scale bar, 100 μm.