Abstract
BackgroundThe prognosis of spinal cord injury (SCI) is closely related to secondary injury, which is dominated by neuroinflammation. There is evidence that α-synuclein aggregates after SCI and that inhibition of α-synuclein aggregation can improve the survival of neurons after SCI, but the mechanism is still unclear. This study was designed to investigate the effects of α-synuclein on neuroinflammation after SCI and to determine the underlying mechanisms.MethodA T3 spinal cord contusion model was established in adult male Sprague-Dawley rats. An SNCA-shRNA-carrying lentivirus (LV-SNCA-shRNA) was injected into the injury site to block the expression of α-synuclein (forming the SCI+KD group), and the SCI and sham groups were injected with an empty vector. Basso-Beattie-Bresnahan (BBB) behavioural scores and footprint analysis were used to detect motor function. Inflammatory infiltration and myelin loss were measured in the spinal cord tissues of each group by haematoxylin-eosin (HE) and Luxol Fast Blue (LFB) staining, respectively. Immunohistochemistry, Western blot analysis, and RT-qPCR were used to analyse protein expression and transcription levels in the tissues. Immunofluorescence was used to determine the morphology and function of glial cells and the expression of matrix metalloproteinase-9 in the central canal of the spinal cord. Finally, peripheral serum cytokine levels were determined by enzyme-linked immunosorbent assay.ResultsCompared with the SCI group, the SCI+KD group exhibited reduced inflammatory infiltration, preserved myelin, and functional recovery. Specifically, the early arrest of α-synuclein inhibited the pro-inflammatory factors IL-1β, TNF-α, and IL-2 and increased the expression of the anti-inflammatory factors IL-10, TGF-β, and IL-4. The neuroinflammatory response was regulated by reduced proliferation of Iba1+ microglia/macrophages and promotion of the shift of M1-polarized Iba1+/iNOS+ microglia/macrophages to M2-polarized Iba1+/Arg1+ microglia/macrophages after injury. In addition, compared with the SCI group, the SCI+KD group also exhibited a smaller microglia/astrocyte (Iba1/GFAP) immunostaining area in the central canal, lower MMP-9 expression, and improved cerebrospinal barrier function.ConclusionLentivirus-mediated downregulation of α-synuclein reduces neuroinflammation, improves blood-cerebrospinal barrier function, promotes functional recovery, reduces microglial activation, and promotes the polarization of M1 microglia/macrophages to an M2 phenotype to confer a neuroprotective immune microenvironment in rats with SCI.
Highlights
Traumatic spinal cord injury (SCI) is caused by an external force applied directly or indirectly to the spinal cord
We designed a total of 5 sets of SNCA-Short hairpin RNA (shRNA) sequence plasmids with green fluorescent protein (GFP) fluorescent tags, integrated them into lentiviruses, and transfected them into HEK293 cells to observe the degree of transfection
During secondary injury after SCI, α-Syn reduced the activation of microglia/astrocytes and the expression of Inducible nitric oxide synthase (iNOS) and promoted the transformation of M1 macrophages to the M2 phenotype, and these changes were accompanied by increases in Arg1 and IL-10 expression
Summary
Traumatic spinal cord injury (SCI) is caused by an external force applied directly or indirectly to the spinal cord. Studies have shown that in the context of SCI, mechanical damage causes neurons and glia at the injury site to die within minutes to hours This death is followed by a delayed secondary injury phase in which the characteristic neuroinflammatory response persists. SCI-induced glial activation and the subsequent release of inflammatory factors such as interleukin (IL), tumour necrosis factor (TNF), and interferon (IFN) accelerate neuronal death while inducing vascular endothelial cells to express multiple cell adhesion and chemotaxis molecules to attract more inflammatory factors These factors typically stimulate nitric oxide (NO) release, increase capillary permeability, and cause blood-cerebrospinal barrier (BCSB) dysfunction [3, 4]. SCI triggers a range of cellular and molecular events, including microglial/astrocyte activation, peripheral bloodderived macrophage infiltration, pro-inflammatory/antiinflammatory response imbalance, abnormal mitochondrial activity, oxidative stress, abnormal protein aggregation, and free radical toxicity These processes induce neuronal death and lead to permanent neurological deficits. This study was designed to investigate the effects of α-synuclein on neuroinflammation after SCI and to determine the underlying mechanisms
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