Ultrarapid Inflammation of the Olfactory Bulb After Spinal Cord Injury: Protective Effects of the Granulocyte Colony-Stimulating Factor on Early Neurodegeneration in the Brain

Abstract

The correlation among olfactory dysfunction, spinal cord injury (SCI), subjective cognitive decline, and neurodegenerative dementia has been established. Impaired olfaction is considered a marker for neurodegeneration. Hence, there is a need to examine if SCI leads to olfactory dysfunction. In this study, the brain tissue of mice with spinal cord hemisection injury was subjected to microarray analysis. The mRNA expression levels of olfactory receptors in the brain began to decline at 8 h post-SCI. SCI promoted neuroinflammation, downregulated the expression of olfactory receptors, decreased the number of neural stem cells (NSCs), and inhibited the production of neurotrophic factors in the olfactory bulbs at 8 h post-SCI. In particular, the SCI group had upregulated mRNA and protein expression levels of glial fibrillary acidic protein (GFAP; a marker of astrocyte reactivation) and pro-inflammatory mediators [IL-1β, IL-6, and Nestin (marker of NSCs)] in the olfactory bulb compared to levels in the sham control group. The mRNA expression levels of olfactory receptors (Olfr1494, Olfr1324, Olfr1241, and Olfr979) and neurotrophic factors [brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor (NGF)] were downregulated in the olfactory bulb of the SCI group mice at 8 h post-SCI. The administration of granulocyte colony-stimulating factor (G-CSF) mitigated these SCI-induced pathological changes in the olfactory bulb at 8 h post-SCI. These results indicate that the olfactory bulb is vulnerable to environmental damage even if the lesion is located at sites distant from the brain, such as the spinal cord. Additionally, SCI initiated pathological processes, including inflammatory response, and impaired neurogenesis, at an early stage. The findings of this study will provide a basis for future studies on pathological mechanisms of early neurodegenerative diseases involving the olfactory bulb and enable early clinical drug intervention.

Keywords: granulocyte colony stimulating factor; neurodegenerative disease; neuroinflammation; olfactory bulb; olfactory dysfunction; spinal cord injury; subjective cognitive decline.

FIGURE 1

Schematic diagram depicting spinal cord injury (SCI)-mediated olfactory dysfunction and the subsequent subjective cognitive decline (SCD)/neurodegenerative dementia. Olfactory dysfunction is an early indicator of neurological diseases, including SCD and neurodegenerative dementia. Previous studies have reported the correlation between SCD and neurodegenerative disease (ND) (indicated as red arrow no. 1). Generally, olfactory impairment is involved in the progression of ND (indicated as red arrow no. 2) or the progression of SCD to ND (from red arrow no. 3 to 1). Thus, SCI promotes ND (indicated as blue arrow no. 4), as well as the progression from initial SCD to ND (from blue arrow no. 5 to red arrow no. 1). This study demonstrated that SCI promotes neuroinflammation in the olfactory bulb at an ultrarapid stage after SCI. Thus, SCI may mediate the pathological mechanisms of neurodegeneration.

FIGURE 2

Spinal cord injury (SCI) can activate the astrocytes in the olfactory bulb at 8 h post-spinal cord hemisection injury in mice. Representative images of GFAP-stained sections of the olfactory bulbs of the sham control (B), SCI (C), SCI + granulocyte colony-stimulating factor (G-CSF) i.p., and SCI + G-CSF oral groups. (A) Schematic illustration of the olfactory bulb (marked with dashed box) of all groups subjected to mRNA, protein, and immunofluorescence analyses. The SCI group exhibited a higher number of GFAP-positive cells [as indicated by arrow in panel (C)] than the sham control group (B). This indicated astrocytic activation and potential astrocyte-mediated inflammatory responses in the olfactory bulb at 8 h post-SCI. The immunofluorescence intensity of GFAP significantly decreased in the SCI + G-CSF i.p. [as indicated by arrow in panel (D)] and SCI + G-CSF oral groups [as indicated by arrow in panel (E)] [(B–C) magnification 400×]. (E) Vertical bars indicate the mean ± standard error of mean) number of GFAP-stained cells in each group (n = 3). ***P < 0.001 and ^###P < 0.001.

FIGURE 3

Neuroinflammation in the mouse olfactory bulb at 8 h post-spinal cord injury (SCI). (A–C) The mRNA expression levels of IL-1β (A), IL-6 (B), and GFAP (C) in the olfactory bulb of the sham control, SCI, SCI + granulocyte colony-stimulating factor (G-CSF) i.p., and SCI + G-CSF oral groups at 8 h post-SCI. Vertical bars indicate the mean ± standard error of the mean (SEM) (n = 6 for each group). ^∗P < 0.05, ^∗∗∗P < 0.001, ##P < 0.01, and ###P < 0.001. (D–F) The protein expression levels of IL-1β (D), IL-6 (E), and GFAP (F) in the olfactory bulb of the four experimental groups at 8 h post-SCI. Representative immunoblots of IL-1β, IL-6, GFAP, and β-actin (internal control) are shown in the upper panel. The lower panel indicates the ratio of target protein band intensity to β-actin protein band intensity relative to the control group (mean ± SEM). G-CSF mitigates SCI-induced neuroinflammation in the olfactory bulb as evidenced by the decreased expression of IL-1β, IL-6, and GFAP. Vertical bars indicate mean ± SEM (n = 6 for each group). ^∗P < 0.05, ^∗∗P < 0.01, and ##P < 0.01.

FIGURE 4

Spinal cord injury (SCI) decreases the expression of olfactory receptors in the olfactory bulb at 8 h post-SCI. The mRNA expression levels of (A) Olfr1494, (B) Olfr1324, (C) Olfr1241, and (D) Olfr979 in the olfactory bulb of the sham control, SCI, SCI + G-CSF i.p., and SCI + G-CSF oral groups. Vertical bars indicate mean ± standard error of mean (n = 6 in each group). ^∗∗P < 0.01, ^∗∗∗P < 0.001, ##P < 0.01, and ###P < 0.001.

FIGURE 5

Spinal cord injury (SCI) downregulates the expression of neurotrophic factors and the number of neural stem cells (NSCs) in the olfactory bulb at 8 h post-SCI. (A–D) The mRNA expression levels of BDNF (A), GDNF (B), NGF (C), and Nestin (NSC marker) (D) in the olfactory bulb of the sham control, SCI, SCI + granulocyte colony-stimulating factor (G-CSF) i.p., and SCI + G-CSF oral groups. (E) The protein expression levels of Nestin in the olfactory bulb of the four experimental groups at 8 h post-SCI. The upper panel shows the immunoblot of Nestin (177 kDa). β-Actin (42 kDa) served as an internal control. The lower panel indicates the ratio of Nestin protein band intensity to β-actin protein band intensity relative to the control group. Vertical bars indicate mean ± standard error of mean for mRNA (A–D) or protein expression (E) [n = 6 in each group for panels (A–E)]. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, #P < 0.05, ##P < 0.01, and ###P < 0.001.