Horseradish peroxidase (HRP) nerve tracing and rat olfactory bulb injury model establishment.
Eight clean grade male SD rats (250 ± 50 g weight) were selected and randomly divided into 2 groups (8 animals/group), namely, the experimental group with dissociated olfactory bulbs and the control group without dissociated olfactory bulbs, and acclimated to the environment for one week before the formal experiment. Each group of rats was injected with 30% HRP in the nasal cavity of both sides at a dose of 50 µL on each side (25 µL/time, 1 h interval), during which the rats' heads were rotated several times to distribute the HRP evenly in the nasal cavity. After 1 d, all rats were weighed, anesthetized with 10% chloral hydrate administered intraperitoneally, and immobilized on a brain stereotaxic apparatus (ZH-C/S, Anhui, China). The skin, subcutaneous tissue and periosteum of the head were incised along the median line of the skull starting from the nasofrontal suture. The left olfactory bulb was exposed with an electric drill under an operating microscope (M220 F12, Leica, Shanghai, China) from 6.7 mm anterior to the fontanelle point and 2 mm lateral to the median suture, and the left olfactory nerve was exposed with an electric drill (WDZ-2000 miniature electric surgical grinding drill, Jingjie, Shanghai, China) at the sieve plate and 2 mm lateral to the median suture in rats, with the right side serving as an internal control. The left olfactory bulb was cut along the vertical median line of the exposed olfactory bulb with a bluntly curved needle (depth about 8 mm), while the undissociated olfactory bulb group was set up as a control group. The cranial opening was closed with bone wax. The surgical area was rinsed with antibiotic aqueous solution and the incision was closed layer by layer. Postoperatively, normal diet and no antibiotics were used.
Olfactory function and HRP nerve tracing assay in rats with olfactory bulb injury model.
The foraging (buried food nuggets) method was used to detect the alteration of olfactory nerve function in each group of rats. Each group of rats was taken for observation at the optimal time point for stem cell treatment (1 week after surgery). Food restriction (mean 0.5 g per rat per day) and free access to water were started 2 days before the test. A 20 cm x 30 cm x 50 cm container was lined with bedding approximately 5 cm thick, and the food pieces were fixed and buried at 4 cm from the surface. The rats were first placed in the container without food pieces for 1 h to adapt to the environment. Afterwards, the rats were randomly placed into the container one by one, and the time required for each rat to find food from the time it was placed into the container was recorded. The rats were judged to be successful in foraging when they held or bit the food pieces with both forelimbs. If the rat failed to find food within 10 min, the experiment was terminated and the recording was restarted from the next experiment. Each rat received 2 tests at 6 h interval, and the average of the 2 test results was taken. Two rats were taken from each group, and the material was taken on the olfactory bulb and olfactory nerve, frozen sections were prepared, stained by adding TMB color developing solution, and the HRP nerve tracing results were observed by microscopy.
Preparation of primary NSCs.
The cerebral cortex of SD rat pups within 6 h of neonate was aseptically isolated, placed in iced DMEM/F12 medium (Gibco, USA), cut into small pieces, digested by trypsin, repeatedly blown to individual cells, and resuspended using stem cell medium (DMEM/F-12 supplemented with 2×10− 5 g/L bFGF, 2×10− 5 g/L EGF, 2% B27) at a 2.5×104/cm2 density spread in cell culture dishes until the density reached 80% when used.
NSCs differentiation.
Primary NSCs were spread in cell culture plates pretreated with non-toxic Poly-D-lysine solution, and NSCs were induced to differentiate into astrocytes with astrocyte medium (DMEM/F-12 supplemented with 10% FBS).
Identification of differentiated astrocytes.
Using immunofluorescence methods, monoclonal antibodies against nestin (mouse anti-rat, proteintech, Wuhan, China), which labels stem cells, and glial fibrillary acidic protein (GFAP), which labels astrocytes, were added to predifferentiated NSCs and post-differentiated astrocytes, respectively, as well as detection antibodies Alexa Flour488 (goat anti-mouse, Vicmed, Xuzhou, Sigma, USA), GFAP) monoclonal antibody (rabbit anti-rat, Sigma, USA), and detection antibodies Alexa Flour488 (goat anti-mouse, Vicmed, Xuzhou, China) and Alexa Flour594 (goat anti-rabbit, Vicmed) to analyze Nestin and GFAP protein expression in different cells.
NSCs treatment.
Sixteen SD rats were randomly divided into 2 groups (8 rats/group). After adaptation to the environment, surgery was performed, and after exposing the left olfactory bulb, 1×106 NSCs cells labeled with Brdu (Proteintech) in 50 µL were injected into the experimental group of rats with dissociated olfactory bulb, and an equal volume of PBS was injected into the control group of rats with dissociated olfactory bulb. The cranial opening was closed with bone wax. The surgical area was rinsed with an aqueous antibiotic solution and the incision was closed layer by layer. Postoperatively, normal diet was used without antibiotics.
Olfactory function assay, NSCs/glial cell identification and migration assay in NSCs-treated rats.
Each group of rats was observed 1 w postoperatively at the optimal time point of NSCs treatment, and the foraging time of each group was calculated to assess the olfactory function of rats. Immunofluorescence analysis of Nestin and GFAP protein expression in frozen sections of olfactory bulb and olfactory nerve in each group of rats to analyze the expression levels of NSCs and glial cells in brain tissues. Immunofluorescence analysis of Brdu fluorescence signal in frozen sections of rat brain tissue in each group to assess the migration of differentiated NSCs in brain tissue.
Statistical treatment.
All experiments were repeated independently no less than three times, and SPSS 18.0 software was used for statistical processing, and the measurement data were expressed as \(\overline{\text{X}}\)±s with a test level of a = 0.05. Normality test and chi-square test were performed, and if the experimental data were not normal, rank sum test was used; one-way ANOVA (one-way ANOVA) was used for normality.