| Objective:To observe expression of amyloid P protein (Aβ) in rats after spinal cord injury (SCI), and effect of decreased Aβ expression by application of gamma secretase inhibitor DAPT on functional recovery, neuronal survival and microglia activation, and to investigate the mechanisms of Aβ in rats model of SCI.Method:Part 1. Totally 128 female SD rats were divided into four groups:mild injury group (SCI-M), moderate injury group (SCI-Mo), severe injury group (SCI-S) and Sham group, with 32 rats in each group. Models of SCI were established by modified Allen’s assay. Enzyme-linked Immunoabsorbent Assay (ELISA) analysis for serum and CSF Aβ protein was performed. Western blot analysis was performed for AP protein in spinal cord tissue. Locomotor functions of all animals were assessed using the Basso Beattie and Bresnahan (BBB) locomotor rating scale. HE staining was used to observe the morphological changes of spinal cord injury. Pearson correlation analysis were performed between Aβ levels in serum/CSF and recovery hind limb function (BBB score). Part 2 Totally 96 female SD rats were divided into three groups:SCI group, DAPT group and sham group, with 32 rats in each group. BBB score were used for evaluation of locomotor functional recovery. Nissl staining was used for observation of motor neuron in anterior horn of spinal cord. Protein levels of Aβ in serum and cerebrospinal fluid (CSF), and serum levels of IL-1, IL-6, TNF-a were determined by ELISA assay. Content of amyloid precursor protein P(APP) and Aβ protein were determined by Western blot. Immunofluorescence test were performed for observation of Aβ and Iba-1 positive cells and co-localization of Aβ and NeuN in spinal cord.Result:Part 1.(1) BBB scores decreased in rats after SCI procedure, and the decrease was proportional to the severity of injury. (2) Serum and cerebrospinal fluid levels of AP protein started to rise at 12h after SCI, with a peak at 3d post injury(dpi) (P<0.05). Elevated levels of Aβ were proportional to the severity of injury in SCI groups. There still existed a significant difference between SCI-S group and sham group of Aβ levels at 28dpi (P<0.05). (3) Expression of Aβ protein in spinal cord tissue were significantly increased at 1dpi and 3dpi (P<0.05), and the increase of Aβ expression were proportional to the severity of injury. (4) Serum and cerebrospinal fluid levels of Aβ protein at 3dpi were negatively to the motor function BBB score at 28dpi in rats. Part2. (1) Motor function of BBB score in DAPT group was significantly higher than SCI group since 14dpi (P<0.05). (2) Necrosis area in spinal cord dorsal white matter and gray matter in DAPT group was significantly smaller than SCI group at both 3dpi and 14dpi by HE staining. (3) Numbers of motor neurons in spinal cord anterior horn in DAPT group were significantly higher than SCI group at both 3dpi and 14dpi by Nissl staining (P<0.05). (4) ELISA assay showed serum and cerebrospinal fluid levels of Aβ protein in DAPT group were significantly lower than SCI group since 1dpi (P<0.05). (5) Western blot showed APP protein in spinal cord tissue in both DAPT group and SCI group were significantly higher than sham group, while Aβ protein in spinal cord tissue in SCI group were significantly higher than both DAPT group and sham group (P<0.05). (6) Immunofluorescence experiments showed numbers of Aβ and Iba-1 positive cells in DAPT group were significantly lower than SCI group (P<0.05), and DAPT decreased co-localization of Ap and NeuN in motor neurons. (7) Serum levels of IL-1, IL-6 and TNF-a in DAPT group were significantly lower than SCI group (P<0.05).Conclusion:(1) Expression of Aβ protein increased after SCI in rat, and the Aβ levels increased in a time-dependent manner, which were proportional to severity of SCI. (2) In rats model of SCI, higher levels of Aβ in serum and cerebrospinal fluid correlated to worse functional recovery, thus detection of serum and cerebrospinal fluid levels of Aβ protein may have certain reference value for evaluation of SCI. (3) Expression of APP protein and deposition of Aβ increased after SCI in rats, which may lead to activation of microglia, release of inflammation cytokines and accelerated neurons death. (4)DAPT effectively reduced expression of Aβ and promoted functional recovery in rats of SCI. The mechanism may be inhibition of y-secretase reduced Aβ-induced activation of microglia and release of inflammation cytokines, which may improve extracellular environment of neurons. |
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