Olfactory Regeneration with Nasally Administered Murine Adipose-Derived Stem Cells in Olfactory Epithelium Damaged Mice.

In this study, we aimed to determine whether nasally administered murine adipose-derived stem cells (ADSCs) could support olfactory regeneration in vivo. Olfactory epithelium damage was induced in 8-week-old C57BL/6J male mice by intraperitoneal injection of methimazole. Seven days later, OriCell adipose-derived mesenchymal stem cells obtained from green fluorescent protein (GFP) transgenic C57BL/6 mice were nasally administered to the left nostril of these mice, and their innate odor aversion behavior to butyric acid was assessed. Mice showed significant recovery of odor aversion behavior, along with improved olfactory marker protein (OMP) expression on both sides of the upper-middle part of the nasal septal epithelium assessed by immunohistochemical staining 14 d after the treatment with ADSCs compared with vehicle control animals. Nerve growth factor (NGF) was detected in the ADSC culture supernatant, NGF was increased in the nasal epithelium of mice, and GFP-positive cells were observed on the surface of the left side nasal epithelium 24 h after left side nasal administration of ADSCs. The results of this study suggest that the regeneration of olfactory epithelium can be stimulated by nasally administered ADSCs secreting neurotrophic factors, thereby promoting the recovery of odor aversion behavior in vivo.

The Impact of Ovariectomy on Olfactory Neuron Regeneration in Mice.

Estrogen has been shown to affect differentiation and proliferation as a mitogen in various neural systems. Olfactory receptor cells are unique within the nervous system, and have the ability to regenerate even after an individual has reached maturity. Olfactory receptor cells also regenerate after experimentally induced degeneration. The purpose of this study is to observe the influence of estrogen depletion induced by ovariectomy on olfactory nerve regeneration. Female mice underwent bilateral ovariectomy at 8 weeks of age and received intraperitoneal administration of methimazole 1 week later. At 2, 4, and 6 weeks after methimazole administration, the olfactory mucosa was analyzed histochemically to determine olfactory epithelium (OE) thickness, olfactory marker protein distribution, and Ki-67 immunoreactivity. Furthermore, 2 weeks after ovariectomy, trkA protein distribution in the OE and nerve growth factor (NGF) levels in the olfactory bulb were determined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. Our results showed that in ovariectomized mice OMP, Ki-67, and trkA-immunopositive cells expression decreased at 2 weeks after methimazole injection, a time point at which regeneration is underway. At this same time point, although NGF production in the olfactory bulb had increased before methimazole administration, no differences were observed between the ovx and control groups. These results suggest that estrogen depletion induces a suppressive effect on regeneration of olfactory neurons, and that estrogen may have a potential use in the treatment of sensorineural olfactory dysfunction.

Effects of Tokishakuyakusan on Regeneration of Murine Olfactory Neurons In Vivo and In Vitro.

Post-upper respiratory tract infection related olfactory dysfunction typically occurs due to neural damage after an upper respiratory tract infection associated with a common cold or influenza. At present, Tokishakuyakusan, a Japanese traditional Kampo medicine, has been found to be effective for post-viral olfactory dysfunction. However, the pharmacodynamics of Tokishakuyakusan in the treatment of post-viral olfactory dysfunction remains unresolved. We investigated the effects of Tokishakuyakusan on the regeneration of olfactory neurons and expression of nerve growth factor (NGF) in neural systems, using in vivo murine studies and in vitro cell culture studies. Eight-week-old BALB/C female mice were fed a pellet diet with or without Tokishakuyakusan. Degeneration of cells in olfactory epithelium was induced by intraperitoneal methimazole injection. Regeneration of olfactory neurons was observed by histological and immunohistochemical procedures. NGF expression in the olfactory bulb was measured by enzyme-linked immunosorbent assay. NGF gene and protein expression were measured using rat primary cultured astrocytes by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We found that olfactory marker protein, Ki-67, and NGF were more highly expressed in the olfactory epithelium during the regeneration period in mice receiving Tokishakuyakusan. In cultured astrocytes, Tokishakuyakusan as well as its individual components, Atractylodes lancea rhizome and Japanese angelica root, increased NGF expression. Screening assays revealed that NGF production was increased by atractylodin and levistolide A, which are ingredients in Atractylodes lancea rhizome and Japanese angelica root, respectively. These results suggest that Tokishakuyakusan promotes regeneration of olfactory neurons by increasing NGF expression in the olfactory bulb.