All animal procedures were approved by the Ethics Committee of The Affiliated Nanhua Hospital. Male Sprague-Dawley (SD) rats was purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China) aged 13–14 weeks (240–270 g) were maintained at 22 °C in a 12-hour light/dark cycle with free access to food and water, except when carrying out the CUMS procedure. Animals were randomly assigned to CUMS or control groups. The experimental groups were kept in isolation. After 48 hours of adaptation, rats were subjected to CUMS procedures consisting of a variety of unpredictable mild stressors. These included swimming in cold water (4 °C) or hot water (45 °C) for five minutes, cage tilting for 24 hours, water deprivation for 24 hours, fasting for 48 hours, shaking for 15 minutes, wet bedding for 24 hours, tail nip for two minutes, and inversion of the light/dark cycle. Subjects received one of these stressors at random each day for 21 consecutive days. The control group was given usual daily care and kept in groups. After the CUMS procedures, rats were subjected to behavioral tests as described below. Rats were then sacrificed by decapitation and the olfactory mucosa was collected.

Behavioral tests were performed after the CUMS procedure to evaluate depressive-like behavior and olfactory function. Each experiment was repeated three times and the average result recorded.

For the sucrose preference test, rats were first adapted to sucrose water before the test. Briefly, a bottle of 1% sucrose water and a bottle of purified water were given to each subject. Bottles were randomly fixed on each cage’s right or left side. The position of the two bottles was swapped 12 hours later. The sucrose preference test was carried out after 24 hours of adaptation. This was performed for 3 hours. The sucrose preference was calculated as: sucrose water consumption/total water consumption.

For the forced swimming test, rats were placed in a beaker filled with water at 25 $\pm$ 1 °C and the depth was set to 20 centimeters. After 6 minutes of adaptation, the immobility time was recorded during 4 minutes.

For the open field test, rats were placed in a square container (100 $\times$ 100 cm). Following adaptation, the number standing upright was recorded during 4 minutes.

The buried food-seeking test was used to assess olfactory function as previously described [14]. Briefly, each subject was food-deprived for 24 hours, then placed at the center of the test cage to search for a 2 g regular food chow pellet buried 8 centimeters beneath the bedding in a randomly chosen corner of the cage. The latency time, defined as the time between placing the rat into the cage and grasping the buried food with its forepaws and/or teeth, was recorded. If animals could not find the food pellet within 600 s, the test was terminated and a score of 600 s was given.

Following deparaffinization and rehydration, tissue slices were stained with hematoxylin for 5 minutes and then immersed five times in 1% acid ethanol. After several washes, slices were stained with eosin for 3 minutes and then dehydrated with graded alcohol and washed in xylene. Images were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

OE was dissected from the olfactory mucosa under a dissecting microscope (SZX7, Olympus, Tokyo, Japan), cut into small pieces, digested in 0.125% Trypsin-ethylenediaminetetraacetic acid (EDTA) (Gibco, Anaheim, CA, USA) for 30 minutes, then centrifuged at 1400 rpm for 5 minutes and the cell pellet collected. To generate neurospheres, cells were resuspended and cultured in Dulbecco’s Modified Eagle’s Medium/Ham’s F 12 nutrient medium (DMEM/F12) supplemented with 50 ng/mL Epidermal Growth Factor (EGF) (PROSPEC, cyt-217, Rehovot, Israel), 50 ng/mL Recombinant Fibroblast Growth Factor 2 (FGF2) (PROSPEC, cyt-218), 1% ITS-X (Gibco, 51500056, USA), 2% B27 (Gibco, Anaheim, CA, USA), and 1% streptomycin/penicillin. The culture medium was changed every 4 days. For the purification of OE progenitor cells, the primary neurosphere was dissociated and the cells cultured again to generate secondary neurospheres. The fourth-generation neurospheres were collected for subsequent experiments. A commercially available Quantitative PCR mycoplasma detection kit was used for mycoplasma testing (Thermo Fisher, #4460623, Fisher, Waltham, MA, USA), no mycoplasma infection was found in the indicated cells. Nestin (a marker for neural stem cells) immunofluorescence staining was used for authentication of progenitor cells.

Purified progenitor cells dissociated from fourth-generation neurospheres were seeded onto poly-L-lysine-coated coverslips. Cells were fixed and washed, blocked for 60 minutes at room temperature, and incubated with primary antibody against nestin (1:1000; #73349, Cell Signaling Technology, Danvers, MA, USA). The samples were then incubated with a fluorescent secondary antibody (1:1000; A23220, Abbkine, Wuhan, Hubei, China) for two hours in the dark at room temperature. Finally, samples were counterstained with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI, D9542, Sigma, MO, USA) and a fluorescence microscope (Olympus, Tokyo, Japan) was used to analyze the cells. Images were processed using Image-Pro plus software (V6.0, Media Cybernetics, Inc., Baltimore, MD, USA).

OE progenitor cells were labeled with 5-Ethynyl-2${}^{\prime }$-Deoxyuridine (EdU) solution (Servicebio, GDP1023, Wuhan, Hubei, China) for four hours and then fixed with paraformaldehyde. After washing, cells were permeabilized with 0.5% Triton-X and then incubated with Apollo solution. DNA was revealed by staining with DAPI. Preparations were analyzed using a fluorescence microscope (Olympus, Tokyo, Japan).

Cells were seeded onto a 96-well plate at 1 $\times$ 10${}^4$ cells per well. Before testing, cells were incubated for two hours at 37 °C with 10% buffer from the Cell Counting kit-8 (C0039, Beyotime, Beijing, China). A microplate reader (Varioskan LUX, Thermo Fisher, Waltham, MA, USA) was used to analyze the absorbance in each well at 450 nm.

Cells were stained with PI (AP101, Multi Sciences, Hangzhou, Zhejiang, China) and Annexin (AP101, Multi Sciences) as per the manufacturer’s instructions. Cell apoptosis was then determined by flow cytometry methods.

Total RNA from OE progenitor cells was extracted using TRIZOL reagent (Sigma-Aldrich, St. Louis, MO, USA). cDNA was synthesized using a commercially available kit (Takara, Kusatsu, Japan). The expression of mRNA was examined using the SYBR Premix EX Taq I kit (Takara, Japan) on a 7300 Plus Real-Time polymerase chain reaction (PCR) System (Thermo Fisher, Waltham, MA, USA). Thermocycling conditions were as follows: an initial 95 °C step for 30 s, followed by 40 cycles at 95 °C for 5 s, and 60 °C for 60 s. GAPDH was used as the internal reference. The primer sequences used are shown in Table 1.

Cells were homogenized using Radioimmunoprecipitation assay buffer (RIPA) buffer containing Phenylmethanesulfonyl fluoride (PMSF). Proteins were subjected to 10% Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred onto Polyvinylidene Fluoride (PVDF) membranes. After blocking for one hour, the membrane was incubated separately with primary antibodies against $\beta$-3 tubulin (1:1000; #5568, Cell Signaling Technology, Danvers, MA, USA) or $\beta$-actin (1:1000; #4970, Cell Signaling Technology). The membranes were then incubated with peroxidase-conjugated secondary antibody. The antigen-antibody-peroxidase complexes were detected with an enhanced chemiluminescence imaging kit (Millipore, Burlington, MA, USA), and the complexes visualized by a chemiluminescence imaging system (Biorad, Hercules, CA, USA).

All data were expressed as the mean $\pm$ Standard Error of Measurement (SEM). SPSS 22 (IBM Corp., Armonk, NY, USA) was used for the statistical analysis. Experiments with two groups were compared by independent-sample t-test. The significance level was set at p 0.05.

A series of tests for depressive-like behavior was performed to evaluate the rat model of depression. As shown in Fig. 1, rats subjected to CUMS procedures exhibited reduced glucose consumption (p = 0.008; Fig. 1A), increased immobility (p = 0.017; Fig. 1B), and decreased body rearing (p = 0.003; Fig. 1C) compared to the control group. Moreover, depressive-like rats showed poorer olfactory function, as demonstrated by the buried food-seeking test (p = 0.007; Fig. 1D). These results indicate the state of depression was correlated with reduced olfactory performance in a rat model.

Fig. 1.

Evaluation of depressive-like behavior and olfactory sensing function. (A) Sucrose preference test. (B) Immobility time recorded in the forced swimming test. (C) The number of rearing in the open field test. (D) Latency time to find food in the buried food-seeking test. n = 10 per group. *p 0.05, **p 0.01 compared to the control group. CON, control group; CUMS, chronic unpredicted mild stress.

OE was isolated from olfactory mucosa, which is composed of the upper OE and the lower lamina propria (Fig. 2A). Neurosphere formation (an aggregated form of neural progenitor cells) is a key feature of neural progenitor cells. OE biopsies were collected for generating neurospheres, which emerged after culturing for 4 days in sphere formation medium (Fig. 2B). For the purification of progenitor cells, primary neurospheres were dissociated and cultured to generate secondary neurospheres. Fourth-generation neurospheres were then collected for the following experiment. Purified progenitor cells derived from fourth-generation neurospheres stained positively for Nestin (a marker of neural progenitor cells) using immunofluorescence (Fig. 2C). These results demonstrate the successful establishment of an in vitro model of OE progenitor cells.

Fig. 2.

Identification of olfactory epithelium (OE) progenitor cells. (A) HE staining of the olfactory mucosa. * indicates the OE portion of olfactory mucosa. Scale bar is 100 µm. (B) Neurosphere formation. Scale bar is 100 µm. (C) Purified progenitor cells derived from fourth-generation neurospheres express nestin. Scale bar is 50 µm. HE, hematoxylin-eosin; DAPI, 4${}^{\prime }$,6-diamidino-2-phenylindole.

EdU labeling was used to evaluate the proliferative potential of purified OE progenitor cells (Fig. 3A). Compared to controls, OE progenitor cells derived from depressive-like rats showed reduced proliferative potential (p 0.001; Fig. 3B). Cell apoptosis was examined by flow cytometry (Fig. 3C). OE progenitor cells derived from rats exposed to CUMS showed increased apoptosis (p = 0.002; Fig. 3D). Furthermore, OE progenitor cells from depressive-like rats showed decreased cell viability, as measured by Cell Counting Kit-8 (CCK8) (p = 0.012; Fig. 3E). These results suggest that OE progenitor cells from depressive-like rats have reduced ability for expansion.

Fig. 3.

Proliferation and survival ability of OE progenitor cells. (A) EdU labeling for the measurement of proliferation potency. Scale bar is 20 µm. (B) Ratio of EdU${}^{+}$/DAPI. (C) Cell apoptosis was analyzed by flow cytometry. (D) Bar graph showing cell apoptosis. (E) Cell viability detected by CCK8 assay. n = 5 per group. *p 0.05, **p 0.01, ***p 0.001 compared to the control group. EdU, 5-Ethynyl-2${}^{\prime }$-Deoxyuridine; CCK8, Cell Counting Kit-8.

To study neuronal differentiation, purified OE progenitor cells were cultured for 3 days in DMEM/F12 supplemented with fetal bovine serum and B27. OE progenitor cells obtained from depressive-like rats showed reduced mRNA expression of $\beta$-3 tubulin using reverse transcription polymerase chain reaction (RT-PCR) (p = 0.03; Fig. 4A), as well as reduced expression of $\beta$-3 tubulin protein using Western blot (p = 0.03; Fig. 4B,C). These results indicate that exposure of rats to CUMS reduces the neuronal differentiation ability of OE progenitor cells.

Fig. 4.

OE progenitor cells derived from depressive-like rats showed reduced potential for neuronal differentiation. (A) Relative mRNA expression of $\beta$-3 tubulin. (B) Representative immunoblotting of $\beta$-3 tubulin. (C) Relative band intensity showing protein expression of $\beta$-3 tubulin. n = 3 per group. *p 0.05 compared to the control group.

To further evaluate the expression of mRNA related to neural stemness, we performed RT-PCR of cultured OE progenitor cells. Compared to the control group, OE progenitor cells obtained from rats exposed to CUMS showed reduced expression of mRNA related to neural stemness (NES: p = 0.002, SOX2: p = 0.04, OLIG2: p = 0.42; Fig. 5).

Fig. 5.

Relative mRNA expression associated with neural stemness in OE progenitor cells. n = 3 per group. *p 0.05, **p 0.01 compared to the control group.