Background

New neurons are generated throughout life in the OE of mammals including humans (Hahn et al., 2005) and mice (Graziadei et al., 1979b; Kondo et al., 2010). Continuous cell generation is primarily achieved by globose progenitor cells (Jang et al., 2014). Progenitor cells also have the potential to reconstitute all OE cell types after injury, with horizontal basal cells having the greatest tissue stem cell potential (Leung et al., 2007; Schnittke et al., 2015; Gadye et al., 2017).

There exist different methods to specifically injure the OE in laboratory rodents, which in turn allows the study of the regenerative process including stem cell activation, neurogenesis, and neuronal differentiation. Methods include removal of the olfactory bulb (OB) (Schwob et al., 1992), olfactory nerve axotomy (Graziadei et al., 1979a; Suzuki and Takeda, 1991), intranasal administration of zinc sulphate (Matulionis, 1975), inhalation of methyl bromide gas (Hurtt et al., 1988), and intraperitoneal (i.p.) injection of olfactotoxic chemicals such as methimazole, colchicine, and dichlobenil (Genter et al., 1995; Suzuki et al.,1998; Bergman and Brittebo, 1999). All these methods induce the destruction of one or several OE cell types followed by detachment of the OE and subsequent regeneration. The mechanism of action differs depending on the method; thus, there are important differences regarding which cell types are affected and how. Noteworthy, viruses such as SARS-Cov-2 can also specifically damage OE cells (Zhang et al., 2020).

Methimazole (1-methyl-2-mercaptoimidazole, also called thiamazole) is used to treat hyperthyroidism (Astwood et al., 1945) and is known to cause a loss of the sense of smell (olfaction) as a side effect of treatment (Cooper, 1999). The OE is a pseudostratified epithelium in which all mature cells and horizontal basal cells are in contact with the basal lamina. In response to i.p. methimazole, several cell types in the OE, such as Bowman’s gland cells, sustentacular cells, and olfactory sensory neurons, display swollen organelles within four hours, which is followed by detachment of cells from the basal lamina (Bergström et al., 2003). After methimazole injury, the OE completely regenerates in approximately one month, and the first olfactory sensory neurons begin to make synaptic contacts with the OB at approximately 1–2 weeks (Suzukawa et al., 2011). Methimazole has been shown to affect young and aged mice differently, on postnatal day 10 compared with 18 months old (Suzukawa et al., 2011).

In rats, i.p. methimazole doses from 25 mg/kg to 300 mg/kg have been evaluated for OE toxicity (Genter et al., 1995). We conducted a pilot study to elucidate the lowest dose suitable for reproducible OE ablation in mice (Håglin et al., 2020). Single injection of either 75 mg/kg or 100 mg/kg resulted in detachment of the entire OE, whereas 50 mg/kg caused incomplete lesioning since there were residual patches of OE cells that appeared relatively unaffected. These results are in accordance with those of previous studies showing that lower doses of i.p. methimazole are insufficient to obtain complete OE lesions in mice (Brittebo, 1995) or rats (Genter et al., 1995). Since 75 mg/kg and 100 mg/kg yielded similar results, we used the lower dose for further studies.

In our hands, methimazole more reliably causes OE ablation than does dichlobenil, and in practice, methimazole is easier to use than zinc sulphate or methyl bromide gas. Our protocol is the first to describe repeated OE ablation–regeneration cycles. By repeating the injury up to three times, we found a gradual decrease in the regenerative potential of the OE, which is accompanied by the progressive accumulation of two different types of metaplasia (Håglin et al., 2020). Thus, repeated ablation–regeneration cycles of the mouse OE following methimazole treatment may be used, for example, to study: i) the exhaustion of the regenerative capacity of neurogenic tissue stem cells, which may mimic aging; ii) the robustness of the re-establishment of a tissue niche when subjected to repeated injury; iii) the plasticity of target neurons in the OB when challenged to re-establish synapses several times over; iv) the role of OE barrier function in protecting against a nasal route of infection as well as uptake of chemical substances to the brain; and v) the roles of different normal and disease variants of genes in these processes since there are many genetically modified mice available for study.