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Additional file 1: Figure S1. SS-31 does not affect the histological morphology and motor function in non-SCI mice. (A) Photographs of spinal cord sections in the Sham and Sham+SS-31 groups stained with antibody MAP2 (green) (scale bar = 25 μm). (B) MAP2 optical density within uninjured spinal cord on day 28. (C) Photographs of spinal cord sections in the respective groups stained with antibody Syn (green)/NeuN (red) (scale bar = 20 μm). (D) Relevant quantitative results for motor neuron-contacting synapse amounts on day 28 after sham surgery. (E) Photographs of mouse footprints on day 28 following sham surgery. Blue: forepaw print; Red: hind paw print. (F, G) Toe dragging (%) and stride length (cm) analyses of mice at 28 days after sham surgery. (H) Longitudinal spinal cord sections from the groups at 28 days after sham surgery were examined via HE dyeing and Masson dyeing (scale bar = 1000 μm). (I) Basso mouse scale (BMS) for the indicated groups at days 0, 1, 3, 7, 14, 21, and 28. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S2. SS-31 attenuates pyroptosis in microglia after SCI. (A) Typical images of immunofluorescence staining for Caspase-1 and microglia colocalization and in the spinal cords of the Sham, SCI, and SCI+SS-31 groups (scale bar= 25μm) (B) Quantified Caspase-1 immunofluorescence data are presented on the right. (C) Typical images of immunofluorescence staining for NLRP3 and microglia colocalization and in the spinal cords of the Sham, SCI, and SCI+SS-31 groups (scale bar= 25μm) (D) Quantified NLRP3 immunofluorescence data are presented on the right. The white arrows indicate Caspase-1 or NLRP3-positive microglia. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S3. CQ treatment does not affect pyroptosis in non-SCI mice. (A-D) Representative double immunofluorescence images for Caspase-1/NeuN and NLRP3/NeuN in spinal cord from Sham and Sham+CQ groups at 3 dpi (scale bar= 25μm). The quantitative integrated densities of Caspase-1 and NLRP3 in each neuron are shown on the graph. (E) Typical images of WB results of Caspase-1, GSDMD-N, NLRP3, NLRP1, ASC, IL-1β, IL-18 in the spinal cord. GAPDH was utilized as a loading control. (F) Quantification results of the pyroptosis-related protein levels from (E) in both groups. The data are shown as the mean ± SEM. ns indicates no significance. Figure S4. SS-31’s functional recovery effect is inhibited by CQ. (A) Photographs of spinal cord sections in the SCI, SCI+SS-31, SCI+SS-31+CQ groups stained with antibody MAP2 (green) (scale bar = 25 μm). Photographs of spinal cord sections in the respective groups stained with Syn (green)/NeuN (red) (scale bar = 20 μm). (B) Relevant quantitative results for motor neuron-contacting synapse amounts on day 28 after SCI. (C) MAP2 optical density within a spinal cord subjected to injury on day 28. (D) Longitudinal spinal cord sections at 28 dpi after SCI stained with HE dyeing and Masson dyeing (scale bar = 1000 μm). (E) The lesion area in injured spinal cord measured from Masson staining. (F) Photographs of mouse footprints on day 28 following spinal cord injury. Blue: forepaw print; Red: hind paw print. (G, H) Toe dragging (%) and stride length (cm) analyses of mice at 28 dpi after SCI. (I, J) Inclined plane test and Basso mouse scale (BMS) for the indicated groups at days 0, 1, 3, 7, 14, 21, and 28. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S5. SS-31 affects the p-cPLA2 expression in glial cells. (A-B) Typical images of immunofluorescence staining for p-cPLA2 and microglia colocalization in the spinal cords of the Sham, SCI, and SCI+SS-31 groups. The white arrow indicates the p-cPLA2 positive microglia. The ratio of p-cPLA2 positive cells to the total is shown on the right. (C-D) Typical images of immunofluorescence staining for p-cPLA2 and oligodendrocyte colocalization in the spinal cords of three groups. The white arrow indicates the p-cPLA2 positive oligodendrocyte. The ratio of p-cPLA2 positive cells to the total is shown on the right. (E-F) Typical images of immunofluorescence staining for p-cPLA2 and astrocyte colocalization in the spinal cords of three groups. The white arrow indicates the p-cPLA2 positive astrocyte. The ratio of p-cPLA2 positive cells to the total is shown on the right. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S6. SS-31 inhibits the activity of cPLA2 and protect the lysosomal enzymes activity in lysosomes. (A) ELISA results showing the activity of cPLA2 in the spinal cord from Sham, SCI, and SCI+SS-31 groups on postoperative day 3. (B-D) ELISA results of the activity of lysosomal enzymes CTSD and NAGLU in lysosomal (B, C) and cytosolic (D, E) fractions extracted from the spinal cord of Sham, SCI, and SCI+SS-31 groups on postoperative day 3. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S7. Pla2g4a overexpression doesn’t affect pyroptosis, autophagy or LMP in non-SCI mice. (A-B) Representative immunofluorescence images of p-cPLA2 and NeuN in the anterior horn areas of spinal cord from Sham, Sham+AAV-Blank, Sham+AAV-Pla2g4a groups after 3 dpi. (scale bar= 25μm). The quantification data were shown on the right. (C) cPLA2 and p-cPLA2 protein levels in the spinal cord from three groups on postoperative day 3. (D-F) Representative double immunofluorescence images for NLRP3/NeuN and Caspase-1/NeuN in spinal cord from three groups at 3 dpi (scale bar= 25μm). The quantitative integrated densities of NLRP3 and Caspase-1 in each neuron are shown on the graph. (G) Typical images of WB analyses of Caspase-1, GSDMD-N, NLRP3, NLRP1, ASC in the spinal cord. GAPDH was utilized as a loading control. (H-J) Representative double immunofluorescence images for p62/NeuN and LC3/NeuN in spinal cord lesions from three groups at 3 dpi (scale bar= 25μm). The number of LC3 II puncta and the quantitative integrated density of p62 in each neuron are shown on the graph. (K) Typical images of WB analyses of p62 and LC3 in the spinal cord. GAPDH was utilized as a loading control. (L) Comparison of the ratio of diffuse CTSL cells in the anterior horn of spinal cord among three groups. (M) Immunofluorescence staining of NeuN and CTSL in the anterior horn of spinal cord from three groups on postoperative day 3 (scale bar= 25μm). (N) Quantification results of the related protein levels from (C), (G) and (K) in the indicated groups. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S8. SS-31 facilitates functional recovery in SCI mice by inhibiting cPLA2 activity. (A, B, D) Photographs of spinal cord sections in the respective groups stained with MAP2 (green) and Syn (green)/NeuN (red). Relevant quantitative results for MAP2 optical density and motor neuron-contacting synapse amounts on day 28 after SCI. (C) Spinal cord longitudinal sections from the groups at 28 days after SCI were examined via HE dyeing and Masson dyeing (scale bar = 1000 μm). (E) Quantitative analyses of Masson-positive lesions within the spinal cord in the corresponding groups. (F) Photographs of mice footprints on day 28 following spinal cord injury. Forepaw print in blue; hindpaw print in red. (G, H) Stride length (cm) and toe dragging (%) analyses of mice at 28 dpi after SCI. (I-J) Inclined plane test and BMS for the corresponding groups at day 0, 1, 3, 7, 14, 21, and 28. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance. Figure S9. SS-31 protected lysosome function by inhibiting p38 signal pathway. (A-D) ELISA results of the activity of lysosomal enzymes CTSD and NAGLU in lysosomal (A-B) and cytosolic (C-D) fractions extracted from the spinal cord of SCI, SCI+SS-31, and SCI+SS-31+AA groups on postoperative day 3. The data are shown as the mean ± SEM. n = 5. *P < 0.05, **P < 0.01. ns indicates no significance.

Central nervous system (CNS) injury is divided into brain injury and spinal cord injury and remains the most common cause of morbidity and mortality worldwide. Previous reviews have defined numerous inflammatory cells involved in this process. In the human body, neutrophils comprise the largest numbers of myeloid leukocytes. Activated neutrophils release extracellular web-like DNA amended with antimicrobial proteins called neutrophil extracellular traps (NETs). The formation of NETs was demonstrated as a new method of cell death called NETosis. As the first line of defence against injury, neutrophils mediate a variety of adverse reactions in the early stage, and we consider that NETs may be the prominent mediators of CNS injury. Therefore, exploring the specific role of NETs in CNS injury may help us shed some light on early changes in the disease. Simultaneously, we discovered that there is a link between NETosis and other cell death pathways by browsing other research, which is helpful for us to establish crossroads between known cell death pathways. Currently, there is a large amount of research concerning NETosis in various diseases, but the role of NETosis in CNS injury remains unknown. Therefore, this review will introduce the role of NETosis in CNS injury, including traumatic brain injury, cerebral ischaemia, CNS infection, Alzheimer’s disease, and spinal cord injury, by describing the mechanism of NETosis, the evidence of NETosis in CNS injury, and the link between NETosis and other cell death pathways. Furthermore, we also discuss some agents that inhibit NETosis as therapies to alleviate the severity of CNS injury. NETosis may be a potential target for the treatment of CNS injury, so exploring NETosis provides a feasible therapeutic option for CNS injury in the future.

Eutrophication-induced coastal hypoxia can result in stressful habitat for marine living resources and cause great economic losses. Nutrient management strategies have been implemented in many coastal systems to improve water quality. However, the outcomes to mitigate hypoxia have been mixed and usually small when only modest nutrient load reduction was achieved. Meanwhile, there has been increasing recognition of climate change impacts on estuarine hypoxia, given estuaries are especially vulnerable to climate change with multiple influences from river, ocean and the atmosphere. Due to the limitation of observational studies and the lack of continuous historical data, long-term oxygen dynamics in response to the changes of external forces are still not well understood. This study utilized a numerical model to quantitatively investigate a century of change of Chesapeake Bay hypoxia in response to varying external forces in nutrient inputs and climate. With intensifying eutrophication since 1950, model results suggest an abrupt increase in volume and duration of hypoxia from 1950s-1960s to 1970s-1980s. This turning point of hypoxia might be related with Tropical Storm Agnes and consecutive wet years with relatively small summer wind speed. During 1985-2016 when the riverine nutrient inputs were modestly decreased, the simulated bottom dissolved oxygen exhibited a statistically significant declining trend of ~0.01 mgL-1yr-1 which mostly occurred in winter and spring. Warming was found to be the dominant driver of the long-term oxygen decline whereas sea level rise had a minor effect. Warming has overcome the benefit of nutrient reduction in Chesapeake Bay to diminish hypoxia over the past three decades. By the mid-21st century, the hypoxic and anoxic volumes are projected to increase by 10-30% in Chesapeake Bay if the riverine nutrient inputs are maintained at high level as in 1990s. Sea level rise and larger winter-spring runoff will generate stronger stratification and large reductions in the vertical oxygen supply to the bottom water. The future warming will lead to earlier initiation of hypoxia, accompanied by weaker summer respiration and more rapid termination of hypoxia. The findings of this study can help guide climate adaptation strategies and nutrient load abatement in Chesapeake Bay and other hypoxic estuaries.