Dopamine Reduced Pyroptosis and Improved Functional Recovery After Spinal Cord Injury

Background: Neuronal loss, demyelination, and an excessive inammatory response accompany the pathogenesis of spinal cord injury (SCI). The inammatory response is promoted by inammasomes in variety diseases. Dopamine is a neurotransmitter that also functions as a regulator in NLRP3 (nucleotide-binding oligomerization domain-like receptor 3) inammasome-dependent neuroinammation. However, the effects and molecular mechanisms underlying the role of dopamine in SCI are little known. Methods:Functional recovery was assessed using Basso Mouse Scale (BMS) and BMS subscore. Histopathologic damage was evaluated by H&E staining. Demyelination was evaluated using immunouorescence staining of myelin basic protein. Neuronal loss was evaluated by immunochemistry staining of NeuN. Pyroptosis was assessed by ow cytometry, western blot, and cell viability and cytotoxicity assays. Results: This study using mice showed that dopamine was signicantly associated with enhanced locomotor recovery after SCI; with a reduction in NLRP3 inammasome activation, pyroptosis, neuron and myelin loss, and histological changes. In vitro data suggested an association between dopamine and suppressed NLRP3 inammasome activation and neuronal pyroptosis, and greater survival of neurons. Conclusion: Thus, dopamine may be a novel and effective approach for improving recovery after SCI. The protein samples of spinal cord tissue and cells were collected and extracted using RIPA lysate buffer China), and measured with a BCA Protein Assay Kit (Beyotime). The samples were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene diuoride membranes. The membranes were blocked with non-fat dry milk and incubated serially with primary antibodies against the following: NLRP3 (1:1000, #15101, Cell Signaling Technology); apoptosisassociated speck-like protein containing a caspase recruitment domain (ASC) (1:1000, #67824, Cell Signaling Technology); caspase-1 (1:1000, #ab138483, Abcam); GSDMD (1:1000, #ab219800, Abcam); IL-1β (1:1000, #12507, Cell Signaling Technology); and IL-18 (1:500, #ab71495, Abcam). The secondary antibodies were goat-anti mouse GAM007, and goat-anti rabbit GAR0072 (both 1:5000, Lianke). The intensity of these bands was measured with enhanced reagents (Perkin Life Sciences, USA). These were analyzed using image analysis software version 3.0 (Fujilm Tokyo, Japan).


Introduction
Spinal cord injury (SCI) is a devastating disease, characterized by prominent paralysis and sensorimotor impairments [1,2]. The pathology of SCI includes phases of both primary and secondary injury [3]. The primary injury constitutes the irreversible mechanical injury, due to direct impact on the spinal cord [4].
Secondary injury results from a complex cascade of molecular events, including neuronal loss [5,6], excessive in ammatory response [7], demyelination [8], and oxidative stress [9]. Mounting evidence suggests that, among these, neuronal loss [10] and excessive in ammatory response [7] are especially grievous, by causing persistent damage and progressive degeneration of the spinal cord. Attenuation of secondary neuronal death [11], neuroin ammation [12], and demyelination [8] may contribute to the recovery of locomotor function.
During injury of the central nervous system, caspase-1 cleaves GSDMD to generate the N-terminal fragment of GSDMD [14,15]. This leads to rupture of the cellular membrane and the release of cellular contents, including the proin ammatory cytokines interleukin (IL)-1β and IL-18 [16]. Pyroptosis contributes to neuronal loss in traumatic brain injury [17], SCI [18], and demyelination in multiple sclerosis [19].
Dopamine is a neurotransmitter in the brain, and serves as a chemical messenger in some peripheral parts of the body [20,21]; it serves as an important bridge between the nervous and immune systems [22]. There is evidence that dopamine is a negative regulator for the activation of the NLRP3 (nucleotidebinding oligomerization domain-like receptor 3) in ammasome [23]. Dopamine can control NLRP3dependent in ammation, including systemic in ammation induced by lipopolysaccharide, neuroin ammation due to neurotoxin, and peritoneal in ammation caused by monosodium urate crystal [20]. In addition, activation of the NLRP3 in ammasome leads to pro-caspase-1 changes to the subunits of caspase-1, which is involved in pyroptosis [14,15]. However, it is not known whether dopamine administration may be protective after SCI, and its molecular mechanisms have not been elucidated.
This study investigated whether dopamine may bene t therapy for SCI. Speci cally, the effects of dopamine administration after SCI were determined, especially changes in neuron and myelin loss, histopathology, and locomotor recovery. The involvement of pyroptosis in the therapeutic effects of dopamine was also explored.

SCI and dopamine administration
All surgical procedures and animal care were approved by the Hangzhou First People's Hospital's Ethics Committee and were conducted in accordance with the Animal Care guidelines of China. Adult female C57BL/6 mice (purchased from Shanghai SLAC Laboratory Animal, 8-10 wk, 20-25 g) were apportioned to receive vehicle and dopamine treatment.
To impose SCI, the mice in dopamine and vehicle groups were anesthetized with 1% sodium pentobarbital (75 mg/kg). After performing a laminectomy of the T10-T11 vertebrae laminae, the exposed spinal cord in dopamine and vehicle groups was compressed using a vascular clamp (30 g force) for 1 minute, based on previous studies [24,25]. Mice in the sham group only underwent laminectomy without crush injury to the spinal cord. After the above procedures, manual urinary bladder emptying was performed (twice a day until recovery).
Immediately after the injury, mice in dopamine group was injected intraperitoneally with dopamine (50 mg/kg, Sigma) and then repeated every 4 hours for 3 days (totally 18 times). Moreover, mice in vehicle group and sham group was injected intraperitoneally with saline and then repeated every 4 hours for 3 days (totally 18 times). The dopamine dosage was chosen based on a previous study [20]. Based on previous study [26], histology evaluation and functional assessment were only performed in dopamine and vehicle groups.

Functional assessment
The evaluation of locomotor recovery was performed with the 9-point Basso Mouse Scale (BMS) and 11points BMS subscore at 1, 3, 7, 14, 21, and 28 days post-operation in an open-eld test [26,27]. The assessment of hindlimb movements and coordination was made by 2 independent observers blinded to the treatment group, and the consensus score was taken (n = 9 mice in each group).

Tissue collection
The experimental mice underwent euthanasia with intraperitoneal injection of an overdose of sodium pentobarbital at 3 days or 28 days post-SCI. At 3 days after injury, spinal cord samples were removed for western blot and ow cytometry (10 and 5 mm, respectively, centered at the lesion epicenter). At 3 days and 28 days after injury, a spinal cord sample (5 mm, centered at the lesion epicenter) was xed in formaldehyde solution and transversely sliced to 4-µm sections for hematoxylin and eosin (H&E) staining and immunostaining. Nine series of sections at 200-μm intervals rostral and caudal to the injury epicenter were picked up on glass slides.

H&E staining
For H&E staining, sections were stained with H&E reagent. The evaluation of histopathologic changes was performed using a 6-point scale determined by injured neurons in the gray matter and damage of tissue structure, as described previously [28]. The score ranged from 0 (no lesion observed) to 6 (large infarction, more than half of the gray matter area). The histologic score from 4 optical elds (high power eld, 450 μm × 325 μm) in each section were averaged to obtain a nal score in a blinded manner (n = 9 sections/mouse; n = 5 mice/group).
For immunochemistry staining, sections were incubated with 0.3% hydrogen peroxide for 10 minutes, washed in PBS, treated with EDTA (ethylenediaminetetraacetic acid) antigen retrieval solution, and then incubated with anti-NeuN (1:100, #ab177487, Abcam) at 4 °C overnight. Subsequently, sections were washed in PBS, the appropriate amount of enzyme labeled sheep anti-mouse IgG polymer (#PV-6002, Beijing Zhongshu Jinqiao Biotechnology) was added, incubated at room temperature for 20 minutes. The appropriate amount of freshly prepared chromogenic solution was added. Sections were visualized with a uorescence microscope (Olympus, Tokyo, Japan). To obtain quantitative measurements of positive cells in the ventral horn, the number of positive cells from the optical eld (high power eld, 225 μm × 162 μm) in the section were analyzed to get the nal data (n = 9 sections/mouse; n = 5 mice/group).
Primary neuronal culture and injury models Primary cortical neurons were cultured as previously described [29]. Brie y, cerebral cortices from embryonic day-14 mice were minced, dissociated with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA), and then passed through a cell strainer. Cells were plated on poly-L-lysine-coated dishes at a density of 1 × 10 6 /mL and subsequently maintained in neuronal basal medium (supplemented with 2% B27, 1% Lglutamine, and 1% penicillin-streptomycin) in a humidi ed incubator (5% CO 2 and 95% air) at 37 °C.
Cells were collected and incubated with GSDMD (1:100, #ab219800, Abcam) for 24 h and then SYTOX Blue dead cell stain (a high-a nity nucleic acid that penetrates only compromised plasma membranes; Molecular Probes, Eugene, OR, USA) at room temperature for 10 minutes to detect the formation of membrane pores. Subsequently, the cells were examined and analyzed using a ow cytometer (BD Biosciences, San Jose, CA, USA).

Cell viability and cytotoxicity assays
Cell viability was determined using a Cell Counting Kit-8 (CCK-8) Detection Assay Kit (#70-CCK805, Lianke). Cells were seeded in plates, pretreated with dopamine (0.25 mM) for 3 hours, and then OGD was performed. Subsequently, CCK-8 solution was added to the culture, and the absorbance at 450 nm was measured and then cell survival rate was calculated.
The release of lactate dehydrogenase (LDH) was determined for cytotoxicity using a detection assay kit (Jiancheng, catalog #A020-1-2). The supernatant in serum-free media was ltered and then transferred to 96-well plates, and incubated with the reaction mixture. Measurement of absorbance at 450 nm was analysed for quanti cation of LDH concentration.

Statistical analyses
All data in this study are expressed as the mean ± standard error of the mean (SEM). The BMS score was analyzed using 2-way repeated-measures analysis of variance (ANOVA) and then Bonferroni's post hoc test. One-way ANOVA and Tukey's post hoc test was used for comparisons with other groups. Differences were considered statistically signi cant at p < 0.05.

Dopamine enhanced behavioral recovery in mice
First, the effects of dopamine on functional locomotor recovery were assessed using the BMS score at 1, 3, 7, 14, 21, and 28 days after SCI. Locomotor assessment at 1, 3, and 7 days after SCI showed no signi cant difference in BMS scores between the dopamine and vehicle groups. The BMS score of dopamine treated mice inclined to signi cantly elevated levels starting at 7 days post-injury and remained consistent up the end of the follow-up (28 days post-injury) compared with vehicle-treated controls (Fig.  1A).Moreover, the BMS subscore of mice in the dopamine -treated group was signi cantly higher compared to that of mice in the vehicle-treated group from 14 days post-injury to 28 days post-injury (Fig.   1B).

Dopamine alleviated the severity of histopathological impairments in mice
The effects of dopamine on histological changes were investigated via H&E staining of the spinal cords of the mice. In the vehicle mice, there was substantial impairment to the spinal cord, with signi cant alteration of the white matter and edema at 3 days( Fig.2A-2C) and 28 days (Fig.2D-2F). The damage was less severe in mice administrated dopamine(p=0.023 for 3 days; p=0.038 for 28 days).

Dopamine decreased neuronal loss in mice
To investigate the effects of dopamine on neuronal loss after SCI, an analysis was performed of NeuN in the spinal cords of the mice, using immunohistochemistry. At 3 days and 28 days post-injury, the number of NeuN-positive cells in the dopamine group was signi cantly higher than that of the vehicle group (p=0.008 for 3 days, Fig.3A-3C; p=0.003 for 28 days, Fig.3D-3F).

Dopamine attenuated demyelination in mice
To evaluate the effect of dopamine on demyelination, the MBP level was studied using immuno uorescence staining of the spinal cord at 28 days post-injury. The MBP immunoreactivity of the dopamine-treated mice was signi cantly higher than that of the vehicle group (p=0.032, Fig.4A-4C). Additionally, the protein levels of GSDMD, IL-1β, and IL-18 were signi cantly higher in the spinal cords of vehicle mice than in the sham group(p<0.001 for GSDMD, p=0.001 for IL-1β, p=0.07 for IL-18, Fig.5E-5F). However, dopamine treatment was associated with lower protein levels of GSDMD, IL-1β and IL-18 compared with the vehicle group(P=0.016 for GSDMD, p=0.008 for IL-1β, p=0.035 for IL-18, Fig.5E-5F).

Dopamine suppressed cell pyroptosis in mice
These results show that administration of dopamine can induce inhibition of pyroptosis of neurons and oligodendrocytes, and suppress neuroin ammation.

Dopamine inhibited the NLRP3 in ammasome activation in mice
To explore further the mechanism of dopamine on pyroptosis, the activation of NLRP3 in ammasome were detected in the spinal cords of mice. Previous studies have showed that the concentration of NLRP3 peaks at 3 days after SCI [30,31]. In the present study, the western blot analysis conducted 3 days after SCI showed that the levels of NLRP3, ASC, and caspase-1 were signi cantly higher in the vehicle group compared with the sham group(p=0.011 for NLRP3, p<0.001 for ASC, p=0.013 for caspase-1, Fig.6A-6B). Nevertheless, dopamine treatment reversed the increased levels of NLRP3, ASC and caspase-1 induced by SCI( p=0.025 for NLRP3, p=0.017 for ASC, p=0.046 for caspase-1, Fig.6A-6B).

Dopamine inhibited pyroptosis and promoted survival in the primary cultured neurons
The western blot analysis revealed that OGD was associated with signi cantly higher levels of GSDMD, relative to the control cells (p=0.006, Fig.7A-7B). Nevertheless, the GSDMD level in the dopamine group was lower than that in the vehicle group(p=0.023, Fig.7A-7B). In addition, the levels of the proin ammatory cytokines IL-1β and IL-18 in the culture supernatant were investigated. The levels of IL-1β and IL-18 were higher in OGD cells compared with the control cells(p=0.003 for IL-1β, Fig.7C; p=0.019 for IL-18, Fig.7D), whereas the levels of IL-1β and IL-18 in the dopamine group prominently lower compared with those in the vehicle group (p=0.018 for IL-1β, Fig.7C; p=0.032 for IL-18, Fig.7D).
To validate the effects of dopamine on pyroptosis and survival after SCI, primary neurons were exposed to OGD to mimic the SCI model in vitro. We detected that the signi cant higher number of double-positive cells of GSDMD and SYTOX Blue staining in the vehicle group compared with the control group ((p=0.004, Fig.7E,7F). However, dopamine treatment was associated with fewer positive cells (p=0.035, Fig.7E,7F).
Subsequently, the effects of dopamine on OGD-induced cell survival rate and neurotoxicity were investigated by CCK-8 and LDH assay, respectively. Compared with the control group, the cell survival rate was signi cantly lower (p<0.001, Fig.7G), and LDH activity markedly higher (p<0.001, Fig.7H), in the vehicle group. However, the cell survival rate was higher (p=0.002, Fig.7G), and LDH activity (p=0.003, Fig.7H) lower in the dopamine group compared to the vehicle group.

Dopamine inhibited NLRP3 in ammasome activation in the primary cultured neurons
To con rm further the mechanisms of dopamine on pyroptosis, the protein levels of NLRP3, ASC, caspase-1 was examined in the primary neurons. The western blot analysis showed that OGD was associated with an evident increase in NLRP3, ASC, and caspase-1, compared with the control group(p=0.015 for NLRP3, p=0.010 for ASC, p=0.048 for caspase-1, Fig.8A-8B). Nevertheless, our results showed that the level of NLRP3, ASC, and caspase-1 was lower in the dopamine group compared to the vehicle group(p=0.049 for NLRP3, p=0.029 for ASC, p=0.041 for caspase-1, Fig.8A-8B).

Discussion
This study showed that SCI induced pyroptosis, in vivo and in vitro. It was also found that dopamine prevented NLRP3 in ammasome activation, inhibited pyroptosis of neurons and oligodendrocytes, and alleviated the expressions of proin ammatory cytokines in vivo and in vitro after SCI. Importantly, the data revealed that dopamine reduced the loss of neuron and myelin, attenuated tissue impairments, and promoted locomotor recovery after SCI. Thus, these results suggest that dopamine has protective effects against SCI.
IL-1β and IL-18 are important factors in released intracellular contents associated with pyroptosis [32]. IL-1β has the detrimental effects not only on tissue integrity after SCI (in terms of glial activation and lesion size), but also on the plasticity of axons [33]. Moreover, IL-1β can inhibit the functional recovery after neural stem cell transplant administered to treat SCI in rats [34], and may downgrade the prognosis of SCI [35]. Importantly, downregulation of IL-1β after traumatic SCI may be potentially protective, for reducing secondary impairments and improving the outcomes [36]. Furthermore, IL-18 levels correlated with the severity of SCI [37]. In the present study, systemic treatment with dopamine after SCI was associated with lower levels of IL-1β and IL-18. This suggests that dopamine may function to control in ammation.
In the present study, after SCI and an increase in proin ammatory cytokines, a substantial loss of neurons and myelin was noted. Neuronal loss and demyelination have been identi ed as key features in secondary injury, and as promising therapeutic targets for improving the outcome after SCI [39,40]. The importance of neuronal death is evident in the progression of SCI, and paralysis after SCI is due to interruption of axons and the failure of neurons to regenerate [41]. Importantly, preserving survival of motor neurons after traumatic SCI can improve functional recovery [42]. In addition, inhibition of demyelination improved recovery in a mouse model of SCI [43]. The present results showed that dopamine after SCI was signi cantly associated with a reduction in neuronal loss and demyelination, and an amelioration of the histopathological outcomes. These ndings suggest that dopamine alleviates SCImediated tissue impairment.
To explore further the mechanism of neuronal loss, demyelination, and changes in proin ammatory cytokines, pyroptosis was analyzed in vivo and in vitro. Pyroptosis is a pattern of cell death, which is consistent with the release of proin ammatory cytokines such as IL-1β and IL-18 [44]. Pyroptosis is closely associated with neuronal loss [45]. Exposure to zinc may cause in ammasome-mediated pyroptosis in olfactory neurons [46]; and 27-hydroxycholesterol contributes to the pathogenesis of neuronal death by inducing pyroptosis [45]. Valproic acid attenuated neuronal impairment that was due to ischemic/reperfusion injury by inhibiting pyroptosis [47]. The present results indicated that SCI induced pyroptosis of neurons and oligodendrocytes. However, dopamine reduced pyroptosis in vivo and in vitro. Moreover, administration of dopamine diminished the loss of neurons and myelin and improved neuronal survival. Altogether, this study showed that dopamine can attenuate SCI-mediated tissue impairment.
To investigate the mechanisms underlying the effect of dopamine on pyroptosis, the protein levels of NLRP3, ASC, caspase-1 in the spinal cord and primary cultured neurons were determined via western blot. Earlier studies have shown that the NLRP3 in ammasome activation can induce pyroptosis [48]. NLRP3 in ammasome activation mediates pyroptosis induced by radiation in bone marrow-derived macrophages [49], and pyroptosis caused by human rhinovirus and in ammation [50]. Moreover, suppression of NLRP3 in ammasome activation was previously reported to control pyroptosis and the in ammatory response [51]. Inhibition of NLRP3 in ammasome activation with MCC950 alleviated iso urane-induced pyroptosis and cognitive impairment in aged mice [52]. Melatonin protects endothelial cell against pyroptosis via the MEG3/miR-223/NLRP3 axis in atherosclerosis [53]. In the present study, we observed that dopamine signi cantly reduced the levels of NLRP3, ASC, and caspase-1. These results further suggest that dopamine-mediated suppression of pyroptosis presumably depends on the alleviation of NLRP3 in ammasome activation.

Conclusions
In summary, this study provides evidence that dopamine after SCI is effective at reducing loss of neurons and myelin, and suppression of in ammation. The effects of dopamine included inhibition of pyroptosis of neurons and oligodendrocytes, reduction of proin ammatory cytokines, increase of neuronal survival, and attenuation of NLRP3 in ammasome activation. Importantly, all of the biological effects of dopamine treatment signi cantly improved locomotor function and protected against secondary tissue damage. These results support the concept that administration of dopamine may be an effective therapeutic strategy for improving recovery after SCI.