Effects of a metalloporphyrinic peroxynitrite decomposition catalyst,ww-85, in a mouse model of spinal cord injury

The aim of the present study was to assess the effect of a metalloporphyrinic peroxynitrite decomposition catalyst, ww-85, in the pathophysiology of spinal cord injury (SCI) in mice. Spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, production of inflammatory mediators, tissue damage and apoptosis. ww-85 treatment (30–300 µg/kg, i.p. 1 h after the SCI) significantly reduced in a dose-dependent manner: (1) the degree of spinal cord inflammation and tissue injury, (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and PARP activation, (4) pro-inflammatory cytokines expression, (5) NF-κB activation and (6) apoptosis. Moreover, ww-85 significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. The results demonstrate that ww-85 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

Microglial Kv1.3 Channels and P2Y12 Receptors Differentially Regulate Cytokine and Chemokine Release from Brain Slices of Young Adult and Aged Mice

Brain tissue damage following stroke or traumatic brain injury is accompanied by neuroinflammatory processes, while microglia play a central role in causing and regulating neuroinflammation via production of proinflammatory substances, including cytokines and chemokines. Here, we used brain slices, an established in situ brain injury model, from young adult and aged mice to investigate cytokine and chemokine production with particular focus on the role of microglia. Twenty four hours after slice preparation, higher concentrations of proinflammatory cytokines, i.e. TNF-α and IL-6, and chemokines, i.e. CCL2 and CXCL1, were released from brain slices of aged mice than from slices of young adult mice. However, maximal microglial stimulation with LPS for 24 h did not reveal age-dependent differences in the amounts of released cytokines and chemokines. Mechanisms underlying microglial cytokine and chemokine production appear to be similar in young adult and aged mice. Inhibition of microglial Kv1.3 channels with margatoxin reduced release of IL-6, but not release of CCL2 and CXCL1. In contrast, blockade of microglial P2Y12 receptors with PSB0739 inhibited release of CCL2 and CXCL1, whereas release of IL-6 remained unaffected. Cytokine and chemokine production was not reduced by inhibitors of Kir2.1 K+ channels or adenosine receptors. In summary, our data suggest that brain tissue damage-induced production of cytokines and chemokines is age-dependent, and differentially regulated by microglial Kv1.3 channels and P2Y12 receptors.     

Chemokines in CNS injury and repair

Recruitment of inflammatory cells is known to drive the secondary damage cascades that are common to injuries of the central nervous system (CNS). Cell activation and infiltration to the injury site is orchestrated by changes in the expression of chemokines, the chemoattractive cytokines. Reducing the numbers of recruited inflammatory cells by the blocking of the action of chemokines has turned out be a promising approach to diminish neuroinflammation and to improve tissue preservation and neovascularization. In addition, several chemokines have been shown to be essential for stem/progenitor cell attraction, their survival, differentiation and cytokine production. Thus, chemokines might indirectly participate in remyelination, neovascularization and neuroprotection, which are important prerequisites for CNS repair after trauma. Moreover, CXCL12 promotes neurite outgrowth in the presence of growth inhibitory CNS myelin and enhances axonal sprouting after spinal cord injury (SCI). Here, we review current knowledge about the exciting functions of chemokines in CNS trauma, including SCI, traumatic brain injury and stroke. We identify common principles of chemokine action and discuss the potentials and challenges of therapeutic interventions with chemokines.     

BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in rats

The pathophysiology of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro‐inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF‐κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro‐inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro‐inflammatory cytokine production in microglia which ultimately promotes functional recovery after SCI.     

Targeting Enolase in Reducing Secondary Damage in Acute Spinal Cord Injury in Rats

Spinal cord injury (SCI) is a complex debilitating condition leading to permanent life-long neurological deficits. The complexity of SCI suggests that a concerted multi-targeted therapeutic approach is warranted to optimally improve function. Damage to spinal cord is complicated by an increased detrimental response from secondary injury factors mediated by activated glial cells and infiltrating macrophages. While elevation of enolase especially neuron specific enolase (NSE) in glial and neuronal cells is believed to trigger inflammatory cascades in acute SCI, alteration of NSE and its subsequent effects in acute SCI remains unknown. This study measured NSE expression levels and key inflammatory mediators after acute SCI and investigated the role of ENOblock, a novel small molecule inhibitor of enolase, in a male Sprague–Dawley (SD) rat SCI model. Serum NSE levels as well as cytokines/chemokines and metabolic factors were evaluated in injured animals following treatment with vehicle alone or ENOblock using Discovery assay. Spinal cord samples were also analyzed for NSE and MMPs 2 and 9 as well as glial markers by Western blotting. The results indicated a significant decrease in serum inflammatory cytokines/chemokines and NSE, alterations of metabolic factors and expression of MMPs in spinal cord tissues after treatment with ENOblock (100 µg/kg, twice). These results support the hypothesis that activation of glial cells and inflammation status can be modulated by regulation of NSE expression and activity. Analysis of SCI tissue samples by immunohistochemistry confirmed that ENOblock decreased gliosis which may have occurred through reduction of elevated NSE in rats. Overall, elevation of NSE is deleterious as it promotes extracellular degradation and production of inflammatory cytokines/chemokines and metabolic factors which activates glia and damages neurons. Thus, reduction of NSE by ENOblock may have potential therapeutic implications in acute SCI.     

Temporal expression profile of CXC chemokines in serum of patients with spinal cord injury

Chemokines, a subclass of cytokine superfamily have both pro-inflammatory and migratory role and serve as chemoattractant of immune cells during the inflammatory responses ensuing spinal cord injury (SCI). The chemokines, especially CXCL-1, CXCL-9, CXCL-10 and CXCL-12 contribute significant part in the inflammatory secondary damage of SCI. Inhibiting chemokine’s activity and thereby the secondary damage cascades has been suggested as a chemokine-targeted therapeutic approach to SCI. To optimize the inhibition of secondary injury through targeted chemokine therapy, accurate knowledge about the temporal profile of these cytokines following SCI is required. Hence, the present study was planned to determine the serum levels of CXCL-1, CXCL-9, CXCL-10 and CXCL-12 at 3–6 h, 7 and 28 days and 3 m after SCI in male and female SCI patients (n = 78) and compare with age- and sex-matched patients with non-spinal cord injuries (NSCI, n = 70) and healthy volunteers (n = 100). ANOVA with Tukey post hoc analysis was used to determine the differences between the groups. The data from the present study show that the serum level of CXCL-1, CXCL-9 and CXCL-10 peaked on day 7 post-SCI and then declined to the control level. In contrast, significantly elevated level of CXCL-12 persisted for 28 days post SCI. In addition, post-SCI expression of CXCL-12 was found to be sex-dependent. Male SCI patients expressed significantly higher CXCL-12 when compared to control and SCI female. We did not observe any change in chemokines level of NSCI. Further, the age of the patients did not influence chemokines expression after SCI. These observations along with SCI-induced CSF-chemokine level should contribute to the identification of selective and temporal chemokine targeted therapy after SCI.     

Beneficial effects of FeTSPP, a peroxynitrite decomposition catalyst, in a mouse model of spinal cord injury

The aim of the present study was to assess the contribution of peroxynitrite formation in the pathophysiology of spinal cord injury (SCI) in mice. To this purpose, we used a peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTSPP). Spinal cord trauma was induced by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of inflammatory mediators, tissue damage, and apoptosis. FeTSPP treatment (10-100 mg/kg, i.p.) significantly reduced in dose-dependent manner 1 and 4 h after the SCI (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and poly-(ADP-ribose) polymerase activation, (4) proinflammmaory cytokines expression, (5) NF-kappaB activation, and (6) apoptosis (TUNEL staining, Bax and Bcl-2 expression). Moreover, FeTSPP significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. Taken together, our results clearly demonstrate that FeTSPP treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma similarly to dexamethasone, a well-known antiinflammatory agent which we have used as positive control.     

Olprinone Attenuates the Acute Inflammatory Response and Apoptosis after Spinal Cord Trauma in Mice

Background

Olprinone hydrochloride is a newly developed compound that selectively inhibits PDE type III and is characterized by several properties, including positive inotropic effects, peripheral vasodilatory effects, and a bronchodilator effect. In clinical settings, olprinone is commonly used to treat congestive cardiac failure, due to its inotropic and vasodilating effects. The mechanism of these cardiac effects is attributed to increased cellular concentrations of cAMP. The aim of the present study was to evaluate the pharmacological action of olprinone on the secondary damage in experimental spinal cord injury (SCI) in mice.

Methodology/Principal Findings

Traumatic SCI is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should be preventable, no effective treatment options currently exist for patients with SCI. Spinal cord trauma was induced in mice by the application of vascular clips (force of 24 g) to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and production of inflammatory mediators, tissue damage, apoptosis, and locomotor disturbance. Olprinone treatment (0.2 mg/kg, i.p.) 1 and 6 h after the SCI significantly reduced: (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation, (4) pro-inflammatory cytokines, (5) NF-κB expression, (6) p-ERK1/2 and p38 expression and (7) apoptosis (TUNEL staining, FAS ligand, Bax and Bcl-2 expression). Moreover, olprinone significantly ameliorated the recovery of hind-limb function (evaluated by motor recovery score).

Conclusions/Significance

Taken together, our results clearly demonstrate that olprinone treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Radiation-induced apoptosis in the neonatal and adult rat spinal cord

This study was designed to characterize radiation-induced apoptosis in the spinal cord of the neonatal and young adult rat. Spinal cords (C2-T2) of 1-, 2- and 10-week-old rats were irradiated with a single dose of 8, 18 or 22 Gy. Apoptosis was assessed histologically according to its specific morphological features or by using the TUNEL assay. Cell proliferation was assessed immunohistochemically using BrdU. Identities of cell types undergoing apoptosis were assessed using immunohistochemistry or in situ hybridization using markers for neurons, glial progenitor cells, microglia, oligodendrocytes and astrocytes. The time course of radiation-induced apoptosis in 1- or 2-week-old rat spinal cord was similar to that in the young adult rat spinal cord. A peak response was observed at about 8 h after irradiation, and the apoptosis index returned to the levels in nonirradiated spinal cords at 24 h. The neonatal rat spinal cord demonstrated increased apoptosis compared to the adult. Values for total yield of apoptosis over 24 h induced by 8 Gy in the neonatal rat spinal cord were significantly greater than that in the adult. Immunohistochemistry studies using Leu7, galactocerebroside, Rip and adenomatous polyposis coli tumor suppressor protein indicated that most apoptotic cells were cells of the oligodendroglial lineage regardless of the age of the animal. No evidence of Gfap or factor VIII-related antigen-positive apoptotic cells was observed, and there was a small number of apoptotic microglial cells (lectin-Rca1 positive) in the neonatal and adult rat spinal cord. In the neonatal but not adult rat spinal cord, about 10% of the apoptotic cells appeared to be neurons and were immunoreactive for synaptophysin. Labeling indices (LI) for BrdU in nonirradiated 1- and 2-week-old rat spinal cord were 20.0 and 16.3%, respectively, significantly greater than the LI of 1.0% in the 10-week-old rat spinal cord. At 8 h after a single dose of 8 Gy, 13.4% of the apoptotic cells were BrdU-positive in 10-week-old rat spinal cord, whereas 62.4 and 44.1% of the apoptotic cells showed BrdU incorporation in 1- and 2-week-old rat spinal cord, respectively. Regardless of the age of the animal, the apoptosis indices in BrdU-positive cells were greater than those in BrdU-negative cells. We conclude that the neonatal spinal cord demonstrates a greater level of apoptosis after exposure to ionizing radiation than the young adult spinal cord. This increase in apoptosis may be associated in part with the greater percentage of proliferating cells in the neonatal spinal cord, which demonstrate a greater level of radiation-induced apoptosis than nonproliferating cells.     

Oncostatin M Reduces Lesion Size and Promotes Functional Recovery and Neurite Outgrowth After Spinal Cord Injury

The family of interleukin (IL)-6 like cytokines plays an important role in the neuroinflammatory response to injury by regulating both neural as well as immune responses. Here, we show that expression of the IL-6 family member oncostatin M (OSM) and its receptor is upregulated after spinal cord injury (SCI). To reveal the relevance of increased OSM signaling in the pathophysiology of SCI, OSM was applied locally after spinal cord hemisection in mice. OSM treatment significantly improved locomotor recovery after mild and severe SCI. Improved recovery in OSM-treated mice was associated with a reduced lesion size. OSM significantly diminished astrogliosis and immune cell infiltration. Thus, OSM limits secondary damage after CNS trauma. In vitro viability assays demonstrated that OSM protects primary neurons in culture from cell death, suggesting that the underlying mechanism involves direct neuroprotective effects of OSM. Furthermore, OSM dose-dependently promoted neurite outgrowth in cultured neurons, indicating that the cytokine plays an additional role in CNS repair. Indeed, our in vivo experiments demonstrate that OSM treatment increases plasticity of serotonergic fibers after SCI. Together, our data show that OSM is produced at the lesion site, where it protects the CNS from further damage and promotes recovery.     

Neuroprotective effect of Scutellaria baicalensis on spinal cord injury in rats

Inflammation has been known to play an important role in the pathogenesis after spinal cord injury (SCI). Microglia are activated after injury and produce a variety of proinflammatory factors such as tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and reactive oxygen species leading to apoptosis of neurons and oligodendrocytes. In this study, we examined the neuroprotective effects of total ethanol extract of Scutellaria baicalensis (EESB) , after SCI. Using primary microglial cultures, EESB treatment significantly inhibited lipopolysaccharide-induced expression of such inflammatory mediators as tumor necrosis factor-α, IL-1β, IL-6, cyclooxygenase-2, and inducible nitric oxide synthase. Furthermore, reactive oxygen species and nitric oxide production were significantly attenuated by EESB treatment. For in vivo study, rats that had received a moderate spinal cord contusion injury at T9 received EESB orally at a dose of 100 mg/kg. EESB inhibited expression of proinflammatory factors and protein carbonylation and nitration after SCI. EESB also inhibited microglial activation at 4 h after injury. Furthermore, EESB significantly inhibited apoptotic cell death of neurons and oligodendrocytes and improved functional recovery after SCI. Lesion cavity and myelin loss were also reduced following EESB treatment. Thus, our data suggest that EESB significantly improve functional recovery by inhibiting inflammation and oxidative stress after injury.     

Secretion profile of human bone marrow stromal cells: Donor variability and response to inflammatory stimuli

Mesenchymal stem cells (MSC) derived from bone marrow are ideal transplants for a variety of CNS disorders and appear to support recovery after injury by secreting therapeutic factors. There is considerable variability in the secretion profile of MSC derived from different donors and it is known that MSC secretion changes in response to inflammatory stimuli, but no comprehensive analysis has been performed to address these issues. Here we show that MSC from seven donors secrete chemokines and cytokines in variable ranges, with some factors showing high variability. Treatment of cultured MSC with pro-inflammatory cytokines or tissue extracts from injured spinal cord resulted in up-regulation of selected cytokines, whereas treatment with an anti-inflammatory cytokine had little effect, indicating that the secretion profile is tightly regulated by environmental challenges. Patterns of up-regulated cytokines were similar in MSC from different donors suggesting a comparable response to inflammatory stimuli.     

Dual regulation of microglia and neurons by Astragaloside IV‐mediated mTORC1 suppression promotes functional recovery after acute spinal cord injury

Inflammation and neuronal apoptosis contribute to the progression of secondary injury after spinal cord injury (SCI) and are targets for SCI therapy; autophagy is reported to suppress apoptosis in neuronal cells and M2 polarization may attenuate inflammatory response in microglia, while both are negatively regulated by mTORC1 signalling. We hypothesize that mTORC1 suppression may have dual effects on inflammation and neuronal apoptosis and may be a feasible approach for SCI therapy. In this study, we evaluate a novel inhibitor of mTORC1 signalling, Astragaloside IV (AS‐IV), in vitro and in vivo. Our results showed that AS‐IV may suppress mTORC1 signalling both in neuronal cells and microglial cells in vitro and in vivo. AS‐IV treatment may stimulate autophagy in neuronal cells and protect them against apoptosis through autophagy regulation; it may also promote M2 polarization in microglial cells and attenuate neuroinflammation. In vivo, rats were intraperitoneally injected with AS‐IV (10 mg/kg/d) after SCI, behavioural and histological evaluations showed that AS‐IV may promote functional recovery in rats after SCI. We propose that mTORC1 suppression may attenuate both microglial inflammatory response and neuronal apoptosis and promote functional recovery after SCI, while AS‐IV may become a novel therapeutic medicine for SCI.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

Rea regulates microglial polarization and attenuates neuronal apoptosis via inhibition of the NF-κB and MAPK signalings for spinal cord injury repair

Inflammation and neuronal apoptosis aggravate the secondary damage after spinal cord injury (SCI). Rehmannioside A (Rea) is a bioactive herbal extract isolated from Rehmanniae radix with low toxicity and neuroprotection effects. Rea treatment inhibited the release of pro-inflammatory mediators from microglial cells, and promoted M2 polarization in vitro, which in turn protected the co-cultured neurons from apoptosis via suppression of the NF-κB and MAPK signalling pathways. Furthermore, daily intraperitoneal injections of 80 mg/kg Rea into a rat model of SCI significantly improved the behavioural and histological indices, promoted M2 microglial polarization, alleviated neuronal apoptosis, and increased motor function recovery. Therefore, Rea is a promising therapeutic option for SCI and should be clinically explored.     

炎性小体在诱导脊髓损伤神经炎症中的作用

脊髓损伤的治疗与康复一直是医学领域的重大难题,尤其是在改善损伤的神经功能方面进展甚微。继发性损伤是造成脊髓损伤后神经功能障碍的主要原因,炎症反应是继发性损伤阶段最重要的病理过程。急性期通过抑制神经炎症来减轻继发性损伤被认为可减轻神经功能损害而达到神经保护作用。炎性小体是一类蛋白质复合体,由模式识别受体中的NLRs家族和PHYIN家族的受体蛋白质作为主要框架组装并命名,常见的炎性小体包括NLRP1、NLRP3、NLRC4(IPAF)、AIM2等。在感染或受到损伤刺激时,炎性小体在细胞质内组装,并激活促炎症蛋白酶胱天蛋白酶1(caspase-1),活化的胱天蛋白酶1一方面促进促炎症细胞因子IL-1β和IL-18的前体成熟和分泌,另一方面介导细胞焦亡。细胞焦亡以细胞肿胀破裂并释放细胞内容物为特征,是在炎症和应激的病理条件下诱导的程序性细胞死亡方式。促炎症细胞因子和焦亡释放的胞内物质都可作为促炎信号引发炎症反应。近期发现,炎性小体通过诱导促炎因子释放以及介导细胞焦亡等途径, 参与激活脊髓损伤后的炎症级联反应,加重继发性神经炎症。靶向抑制炎性小体的激活可减轻炎症反应,促进神经细胞存活,达到神经保护作用。因此,炎性小体有望成为脊髓损伤治疗的新靶点。本文拟从炎性小体的结构及其在脊髓损伤中的作用、激活机制和治疗前景进行综述,以期为后续研究提供思路。