Decreased Brain Water Content Research Articles

The therapeutic value of adipose-derived pericyte transplantation after intracerebral hemorrhage in rats.

Intracerebral hemorrhage (ICH) is a devastating cerebrovascular associated with inflammation and BBB disruption. Pericytes plays a critical role in neurological diseases, while whether pericytes could be utilized to treat ICH remains to be elucidated. Here, we isolated CD146+CD34- pericytes from rat adipose tissues (ADPs). Fluorescence-activated cells maintained their cell morphology and differentiation potential and expressed pericytes markers (CD146, NG2, and PDGFRβ) but not endothelial markers (CD31, CD34, and CD45). ADPs transplantation improved the neuro-behavioral functions in ICH rats and resulted in decreased hematoma volume and neuron loss after ICH. Besides, ADPs graft restrained the infiltration of neutrophils and reactive microgliosis after ICH injury around the peri-hematoma area of rats, as evidenced by increased Iba1- and MPO immunoreactivity. The transplanted pericytes were covered on endothelial cells, and promoted angiogenesis and vascular basement membrane formation in the peri-hematoma area of ICH rats, as shown by double staining of PDGFRβ and CD31/CollagenIV. The decreased brain water content and Evans Blue leakage proved the protective role of ADPs graft on BBB permeability. Finally, transplanted ADPs increased the expression of VE-cadherin, ZO-1, and claudin-5, leading to stable endothelial cell-cell adhesion and tight junction. In conclusion, the transplantation of APDs improved neuronal after ICH, which involved different mechanisms including neuroinflammation regulation and BBB dysfunction recovery. Our results supported that ADPs might be the ideal cell type for ICH therapy and provided insights into the potential cell therapy for further ICH treatment.

Sodium tanshinone IIA sulfonate attenuates sepsis-associated brain injury via inhibiting NOD-like receptor 3/caspase-1/gasdermin D-mediated pyroptosis

Sodium tanshinone IIA sulfonate (STS) has been reported to protect organ function in sepsis. However, the attenuation of sepsis-associated brain injury and its underlying mechanisms by STS has not been established. C57BL/6 mice were used to establish the cecal ligation perforation (CLP) model, and STS was injected intraperitoneally 30min before the surgery. The BV2 cells were stimulated by lipopolysaccharide after being pre-treated with STS for 4h. The STS protective effects against brain injury and in vivo anti-neuroinflammatory effects were investigated using the 48-hour survival rate and body weight changes, brain water content, histopathological staining, immunohistochemistry, ELISA, RT-qPCR, and transmission electron microscopy. The pro-inflammatory cytokines of BV2 cells were detected by ELISA and RT-qPCR. At last, the levels of NOD-like receptor 3 (NLRP3) inflammasome activation and pyroptosis in brain tissues of the CLP model and BV2 cells were detected using western blotting. STS increased the survival rate, decreased brain water content, and improved brain pathological damage in the CLP models. STS increased the expressions of tight junction proteins ZO-1 and Claudin5 while reducing the expressions of tumor necrosis factor α (TNF-α), interleukin-1β(IL-1β), and interleukin-18 (IL-18) in the brain tissues of the CLP models. Meanwhile, STS inhibited microglial activation and M1-type polarization in vitro and in vivo. The NLRP3/caspase-1/ gasdermin D (GSDMD)-mediated pyroptosis was activated in the brain tissues of the CLP models and lipopolysaccharide (LPS)-treated BV2 cells, which was significantly inhibited by STS. The activation of NLRP3/caspase-1/GSDMD-mediated pyroptosis and subsequent secretion of proinflammatory cytokines may be the underlying mechanisms of STS against sepsis-associated brain injury and neuroinflammatory response.

Dexmedetomidine promotes the functional recovery of mice after acute ischemic stroke via activation of the a2-adrenoceptor.

Ischemic stroke (IS) is a well-known acute cerebrovascular disease characterized by high disability, morbidity, and recurrence rates with no effective treatments. Dexmedetomidine (DEX), a selective a2-adrenoceptor agonist used in anaesthesiology and pain management, has been found to exhibit neuroprotective effects in various diseases. However, its role in IS and the underlying mechanisms remains to be determined. Hence, the aim of the present study was to investigate the neuroprotective role of DEX in the recovery of mice following middle cerebral artery occlusion (MCAO). Mice were used to establish the animal model, and then DEX was injected. Behavioural tests (neurological function assessments, grip test, and rotarod test), brain water content measurement, ELISA, and measurement of oxidative stress were performed. DEX activated a2-adrenoceptor and resulted in reduced brain injury, as indicated by the decreased brain water content, S100 Calcium Binding Protein B (S100B) content, and neuron-specific enolase (NSE) content, whilst also inhibiting oxidative stress, as indicated by the increased total antioxidant capacity, catalase, glutathione, and superoxide dismutase levels, and decreased malondialdehyde and glutathione oxidized levels. Neuroinflammation was also reduced as indicated by the decrease in IFN-g, IL-1a, IL-1b, IL-6, TNF-a, and MMP levels, improved the recovery of neurological function, as indicated by the decreased neurological function score and mNSS, and increased grip strength and rotarod performance in MCAO mice. These combined results suggest that DEX may be a novel strategy for the treatment of IS.

Salvianolic acids for injection alleviates cerebral ischemia-induced neurodegeneration by inhibiting endoplasmic reticulum stress and neuroinflammation

BackgroundCerebral ischemia is one of the leading causes of neurological deficit, dementia, and disability globally. Salvianolic acids for injection (SAFI), a multi-component drug derived from Salvia miltiorrhiza L., has been clinically used in China to treat ischemic stroke. PurposeThis study was designed to investigate the mechanisms of SAFI in alleviating cerebral ischemia-induced neurodegeneration, especially its regulatory effects on excessive ER stress. Basic proceduresThe rat acute cerebral ischemic injury model was established by a 1.5 h occlusion of the middle cerebral artery followed by 24 h of reperfusion. The neurological deficits, neurobehavior, cerebral infarct volume and edema of rats were examined. Hematoxylin and eosin staining, transmission electron microscopy analysis and TUNEL staining were performed to study the brain injury and cell apoptosis. Western blot and immunofluorescence assay were carried out to study the effects and mechanism of SAFI on ischemic injury. Main findingsResults showed that SAFI significantly inhibited the acute neurodegeneration, reduced brain infarct volumes and decreased brain water content of rats. SAFI also downregulated the relative protein levels of Bax/Bcl-2, Bim, Caspase-12, and reduced the number of TUNEL-positive cells in brain tissue, indicating the anti-apoptotic effects of SAFI. Both of immunofluorescence assay and western blot results showed that SAFI downregulated the expression of endoplasmic reticulum (ER) stress maker GRP78. SAFI dramatically inhibited the ER stress-related PERK/eIF2α/ATF4/CHOP and IRE1α/TRAF2/ASK1/JNK signaling pathways. SAFI also alleviated NF-κB triggered inflammatory response, and inhibited the production of IL-6 and IL-β. ConclusionsOur studies indicated that inhibition of excessive ER stress contributes to the protective effects of SAFI against cerebral ischemia-induced neurodegeneration and neuroinflammation.

2-Methoxyestradiol Alleviates Neuroinflammation and Brain Edema in Early Brain Injury After Subarachnoid Hemorrhage in Rats.

ObjectiveNumerous studies have shown that neuroinflammation and brain edema play an important role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). 2-Methoxyestradiol (2-ME) has been shown to have anti-inflammatory and anti-angiogenic effects. This study aimed to investigate the effects of 2-ME on neuroinflammation and brain edema after SAH and its underlying mechanism of action.MethodsRats were used to produce an endovascular puncture model of SAH. 2-ME or the control agent was injected intraperitoneally 1 h after SAH induction. At 24 h after surgery, the neurological score, SAH grading, brain water content, and blood–brain barrier (BBB) permeability were examined. The microglial activation level in the rat brain tissue was determined using immunofluorescence staining, whereas the cell apoptosis in the rat brain tissue was assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, the levels of Interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α were measured by enzyme linked immunosorbent assay, and the expression levels of ZO-1, occludin, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and matrix metallopeptidase (MMP)-9 in the rat brain tissue were determined using western blotting.ResultsTwenty-four hours after SAH, brain water content, BBB permeability, microglial activation, and cell apoptosis were significantly increased, whereas neurological function deteriorated significantly in rats. Treatment with 2-ME significantly decreased brain water content, BBB permeability, microglial cell activation, and cell apoptosis and improved neurological dysfunction in rats. Treatment with 2-ME reduced the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α), which were significantly elevated 24 h after SAH. Treatment with 2-ME alleviated the disruption of tight junction proteins (ZO-1 and occludin), which significantly decreased 24 h after SAH. To further determine the mechanism of this protective effect, we found that 2-ME inhibited the expression of HIF-1α, MMP-9, and VEGF, which was associated with the inflammatory response to EBI and BBB disruption after SAH.Conclusion2-ME alleviated neuroinflammation and brain edema as well as improved neurological deficits after SAH in rats. The neuroprotective effect of 2-ME on EBI after SAH in rats may be related to the inhibition of neuroinflammation and brain edema.

Shape effect of cerium oxide nanoparticles on mild traumatic brain injury

The catalytic performance and therapeutic effect of nanoparticles varies with shape. Here, we investigated and compared the therapeutic outcomes of ceria nanospheres (Ceria NSs) and ceria nanorods (Ceria NRs) in an in vivo study of mild traumatic brain injury (mTBI). In vivo TBI was induced in a mouse model of open head injury using a stereotaxic impactor. Outcomes including cytoprotective effects, cognitive function, and cerebral edema were investigated after retro-orbital injection of 11.6 mM of ceria nanoparticles. Ceria nanoparticles significantly reduced fluoro-jade B (FJB)-positive cells and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, and restored mRNA levels of superoxide dismutase 1 (SOD1) and SOD2. They also decreased the cyclooxygenase-2 (COX-2) expression compared with the untreated control group. Comparing the two nanomaterials, Ceria NRs showed less stable and high-energy (100) and (110) planes, which increased the number of active sites. The Ce3+/Ce4+ molar ratio of Ceria NRs (0.40) was greater than that of Ceria NSs (0.27). Ceria NRs (0.059 ± 0.021) appeared to exhibit better anti-inflammatory effect than Ceria NSs (0.133 ± 0.024), but the effect was statistically insignificant (p = 0.190). Ceria nanoparticles also improved cognitive impairment following mTBI compared with the control group, but the effect did not differ significantly according to the nanoshape. However, Ceria NRs (70.1 ± 0.5%) significantly decreased brain water content compared with Ceria NSs (73.7 ± 0.4%; p = 0.0015), indicating a more effective reduction in brain edema (p = 0.0015). Compared with Ceria NSs, the Ceria NRs are more effective in alleviating cerebral edema following in vivo mTBI.

Effect of Tongfu Xingshen capsule on the endogenous neural stem cells of experimental rats with intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) can stimulate neural regeneration, promoting tissue repair and recovery of nerve function. Tongfu Xingshen capsule (TXC) is a Chinese medicinal formula used to treat ICH and has been shown to protect brain tissue and improve nerve function in clinical studies. However, the effect of TXC on endogenous neural stem cells (NSCs) remains elusive. To explore the mechanisms underlying TXC action, a rat model of ICH was established. The effects of TXC on the proliferation and differentiation of NSCs were assessed in the subventricular zone (SVZ). TXC significantly improved nerve function defects, decreased brain water content and restored blood-brain barrier integrity. Additionally, BrdU labeling showed that both high and low doses of TXC significantly increased the proportion of actively cycling NSCs positive for Nestin and glial fibrillary acidic protein, but did not affect the proliferation rates of NeuN-positive neurons. Finally, TXC also upregulated the mRNA levels of brain-derived neurotrophic factor and its receptor, TrκB, in affected brain tissues. Taken together, TXC accelerated neural repair and functional recovery after brain injury by potentially enhancing the proliferation and differentiation of endogenous NSCs into astroglial cells in the SVZ area.

Extracellular vesicles derived from bone marrow mesenchymal stem cells alleviate neuroinflammation after diabetic intracerebral hemorrhage via the miR-183-5p/PDCD4/NLRP3 pathway.

Intracerebral hemorrhage (ICH) induced by diabetes results in further brain injury and nerve cell death. Bone marrow mesenchymal stem cell (BMSC) transplantation contributes to attenuating neurological deficits after ICH. This study investigated the mechanism of extracellular vesicles (EVs) derived from BMSCs in reducing neuroinflammation after diabetic ICH. BMSC-EVs were isolated and identified. The rat model of db/db-ICH was established and the model rats were administered with EVs. miR-183-5p expression in brain tissues of db/db-ICH rats was detected. The brain injury of db/db-ICH rats was evaluated by measuring neurobehavioral score, brain water content and inflammatory factors. BV2 cells were cultured in vitro to establish high-glucose (HG)-Hemin-BV2 cell model. The levels of reactive oxygen species (ROS) and inflammatory factors in BV2 cells were measured, and BV2 cell viability and apoptosis were assessed. The targeting relationship between miR-183-5p and PDCD4 was predicted and verified. The activation of PDCD4/NLRP3 pathway in rat brain tissues and BV2 cells was detected. miR-183-5p expression was reduced in db/db-ICH rats brain tissues. BMSC-EVs ameliorated cranial nerve function, decreased brain water content and repressed inflammatory response by carrying miR-183-5p. BMSC-EVs mitigated HG-Hemin-BV2 cell injury, reduced ROS level and suppressed inflammatory response. miR-183-5p targeted PDCD4. PDCD4 promoted BV2 cell inflammation by activating the NLRP3 pathway. BMSC-EVs inhibited HG-Hemin-BV2 cell inflammation through the miR-183-5p/PDCD4/NLRP3 pathway, and inhibition of miR-183-5p reversed the protective effect of EVs. BMSC-EVs carried miR-183-5p into db/db-ICH rat brain tissues and repressed the NLRP3 pathway by targeting PDCD4, thus alleviating neuroinflammation after diabetic ICH.

Isorhynchophylline Relieves Ferroptosis-Induced Nerve Damage after Intracerebral Hemorrhage Via miR-122-5p/TP53/SLC7A11 Pathway.

Isorhynchophylline (IRN), a component of traditional Chinese herb Uncaria rhynchophylla, possesses strong antioxidant activity. Ferroptosis induced by iron overload causes cell oxidative stress after intracerebral hemorrhage (ICH). Therefore, this study aims to explore the effects of IRN on the ferroptosis following ICH. In this study, mouse hippocampal HT-22 cells were treated with ferric ammonium citrate (FAC) alone or together with IRN, and we found IRN reduced the FAC-induced cell damage. Then, cells were treated with IRN following treatment with FAC after transfection with miR-122-5p inhibitor, and the results showed IRN reduced the FAC-induced decrease of miR-122-5p levels and relieved the ferroptosis by detecting ferroptotic marker proteins, iron ion concentration and oxidative stress level; after transfection with miR-122-5p inhibitor, the protective effects of IRN against FAC-induced ferroptosis in these cells were weakened. TP53 (also known as p53) was verified as a target of miR-122-5p by using dual luciferase reporter assay, and restoration of TP53 attenuated the effects of miR-122-5p on ferroptotic marker proteins expression, iron ion concentration and lipid ROS levels, as well as solute carrier family seven member 11 (SLC7A11) mRNA expression. SLC7A11 siRNA reversed the inhibitory effects of IRN on FAC-induced ferroptosis and oxidative stress levels. Subsequently, IRN increased the mNSS score, and decreased brain water content and EB content in ICH model. Moreover, IRN decreased ferroptosis and lipid ROS level, upregulated the expression of miR-122-5p and SLC7A11 mRNA, and inhibited TP53 expression. Our findings reveal that IRN protects neurocyte from ICH-induced ferroptosis via miR-122-5p/TP53/SLC7A11 pathway, which may provide a potential therapeutic mechanism for ICH.

Recombinant CCL17-dependent CCR4 activation alleviates neuroinflammation and neuronal apoptosis through the PI3K/AKT/Foxo1 signaling pathway after ICH in mice

BackgroundIntracerebral hemorrhage (ICH), a devastating subtype of stroke, is associated with high mortality and morbidity. Neuroinflammation is an important factor leading to ICH-induced neurological injuries. C-C Chemokine Receptor 4 (CCR4) plays an important role in enhancing hematoma clearance after ICH. However, it is unclear whether CCR4 activation can ameliorate neuroinflammation and apoptosis of neurons following ICH. The aim of the present study was to examine the effects of recombinant CCL17 (rCCL17)-dependent CCR4 activation on neuroinflammation and neuronal apoptosis in an intrastriatal autologous blood injection ICH model, and to determine whether the PI3K/AKT/Foxo1 signaling pathway was involved.MethodsTwo hundred twenty-six adult (8-week-old) male CD1 mice were randomly assigned to sham and ICH surgery groups. An intrastriatal autologous blood injection ICH model was used. rCCL17, a CCR4 ligand, was delivered by intranasal administration at 1 h, 3 h, and 6 h post-ICH. CCL17 antibody was administrated by intraventricular injection at 1 h post-ICH. C021, a specific inhibitor of CCR4 and GDC0068, an AKT inhibitor were delivered intraperitoneally 1 h prior to ICH induction. Brain edema, neurobehavioral assessments, western blotting, Fluoro-Jade C staining, terminal deoxynucleotidyl transferase dUTP nick end labeling, and immunofluorescence staining were conducted.ResultsEndogenous expression of CCL17 and CCR4 were increased following ICH, peaking at 5 days post-induction. CCR4 was found to co-localize with microglia, neurons, and astrocytes. rCCL17 treatment decreased brain water content, attenuated short- and long-term neurological deficits, deceased activation of microglia/macrophages and infiltration of neutrophils, and inhibited neuronal apoptosis in the perihematomal region post-ICH. Moreover, rCCL17 treatment post-ICH significantly increased the expression of CCR4, PI3K, phosphorylated AKT, and Bcl-2, while Foxo1, IL-1β, TNF-α, and Bax expression were decreased. The neuroprotective effects of rCCL17 were reversed with the administration of C021 or GDC0068.ConclusionsrCCL17-dependent CCR4 activation ameliorated neurological deficits, reduced brain edema, and ameliorated neuroinflammation and neuronal apoptosis, at least in part, through the PI3K/AKT/Foxo1 signaling pathway after ICH. Thus, activation of CCR4 may provide a promising therapeutic approach for the early management of ICH.

Artesunate Provides Neuroprotection against Cerebral Ischemia-Reperfusion Injury via the TLR-4/NF-κB Pathway in Rats.

Inflammation has an important role in ischemia-reperfusion (I/R) injury. Artesunate (ART) has anti-microbial and anti-inflammatory pharmacological activities, and it is used for various types of serious malaria, including cerebral malaria. ART maintains a high concentration in the brain but little is known about the neuroprotective effect of ART against brain I/R injury. We studied the neuroprotection of ART against brain I/R injury and its underlying mechanism. In this study, rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h. After 24 h of reperfusion, neurological deficits, cerebrum water content, infarct volume, hematoxylin-eosin (H&E)-staining, myeloperoxidase (MPO) activity, and proinflammatory cytokine levels were measured. Administration of 20, 40, 80, and 160 mg/kg ART intraperitoneally (i.p.) 10 min after MCAO significantly decreased brain water content and improved neurological deficits in a dose-dependent manner. An 80 mg/kg dosage was optimal. ART significantly reduced infarct volume, suppressed MPO activity and diminished the expressions of toll-like receptor (TLR)-4, MyD88, nuclear factor-κB (NF-κB), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in the area of the ischemic cortex. The neuroprotective action of ART against focal cerebral I/R injury might be due to the attenuation of inflammation through the TLR-4/NF-κB pathway.

The effects of thymoquinone on memory impairment and inflammation in rats with hepatic encephalopathy induced by thioacetamide.

Hepatic encephalopathy (HE) is a prevalent complication of the central nervous system (CNS) that is caused by acute or chronic liver failure. This study was designed to evaluate the effects of thymoquinone (TQ) on thioacetamide (TAA)-induced HE in rats, and determine the consequential behavioral, biochemical, and histological changes. HE was induced in male Wistar rats by intraperitoneal (i.p.) injection of 200mg/kg TAA once every 48h for 14 consecutive days. Control groups received the normal saline containing 5 % DMSO. Thymoquinone (5, 10, and 20mg/kg) was administered for ten consecutive days intraperitoneally (i.p.) after HE induction and it was continued until the end of the tests. Then, the passive avoidance memory, extracellular single unit, BBB permeability, and brain water content were evaluated. Moreover, hippocampal tissues were used for evaluation of oxidative stress index, inflammatory biomarkers, and histological parameters following HE. As result of the treatment, TQ improved passive avoidance memory, increased the average number of simultaneous firing of spikes/bins, improved the integrity of BBB, and decreased brain water content in the animal model of HE. Furthermore, the results indicated that treatment with TQ decreased the levels of inflammatory cytokines (TNF-α and IL-1β) but increased the levels of glutathione (GSH) and anti-inflammatory cytokine (IL-10) of the surviving cells in the hippocampal tissues. This study demonstrates that TQ may have beneficial therapeutic effects on cognitive, oxidative stress, neuroinflammatory, and histological complications of HE in rat.

Neuroprotective and Anti-inflammatory Effect of Tangeretin Against Cerebral Ischemia-Reperfusion Injury in Rats.

The neuro-inflammation is well known to be an inflammatory response in the brain tissue. Anti-inflammatory therapy in ischemia/reperfusion (I/R) pathogenesis is a potential therapeutic strategy for post-I/R injury. Currently, we made attempt to scrutinize the neuro-protective effect of tangeretin against I/R injury in the brain of experimental rats. I/R injury is induced in the brain via transient middle cerebral artery occlusion (2h) and reperfusion (20h). The infarction area, brain water content and neurofunctional parameters were also estimated. Inflammatory cytokines and brain injury markers were scrutinized at the end of the study. mRNA expression of interleukin 6 (IL-6), toll-like receptor-4 (TLR-4), interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), interferon-γ (IFNG-γ), and transforming growth factor-β1 (TGF-β1) was estimated using the qRT-PCR. Tangeretin significantly (P < 0.001) decreased brain water content, infarct volume, neurological score, brain edema, and Evans blue leakage. Tangeretin significantly (P < 0.001) down-regulated the inflammatory and pro-inflammatory cytokines and oxidative stress parameters in the serum and brain tissue of experimental rats. qRT-PCR data demonstrated that rats treated with tangeretin could significantly (P < 0.001) suppress the IL-1β, TLR-4 TNF-α, IFNG-γ, and IL-6 and boost the expression of TGF-β1 compared with I/R injury rats. The result clearly showed tangeretin neuro-protective and anti-inflammatory effect against I/R injury in rats through suppressed inflammatory reaction.

Targeting CCL20 inhibits subarachnoid hemorrhage-related neuroinflammation in mice.

Recent evidence suggests that CC chemokine ligand 20 (CCL20) is upregulated after subarachnoid hemorrhage (SAH). Here, we investigated the functions of CCL20 in SAH injury and its underlying mechanisms of action. We found that CCL20 is upregulated in an SAH mouse model and in cultured primary microglia and neurons. CCL20-neutralizing antibody alleviated SAH-induced neurological deficits, decreased brain water content and neuronal apoptosis, and repressed microglial activation. We observed increased levels of CCL20, CC chemokine receptor 6 (CCR6), interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), as well as of microglial activation in microglia treated with oxyhemoglobin (OxyHb). CCL20 or CCR6 knockdown reversed the effects of OxyHb on microglia. Conditioned medium from OxyHb-treated microglia induced neuronal apoptosis, while the percentage of apoptotic neurons in the conditioned medium from microglia transfected with CCL20 siRNA or CCR6 siRNA was decreased. We observed no decrease in OxyHb-induced apoptosis in CCL20-knockdown neurons. Conditioned medium from OxyHb-treated neurons led to microglial activation and induced CCR6, IL-1β and TNF-α expression, while CCL20 knockdown in neurons or CCR6 knockdown in microglia reversed those effects. Our results thus suggest CCL20 may be targeted to elicit therapeutic benefits after SAH injury.

MCC950 Inhibits NLRP3 Inflammasome and Alleviates Axonal Injures in Early Stages of Diffuse Axonal Injury in Rats.

Increasing evidence has revealed that neuroinflammation plays a pivotal role in axonal injures. Nucleotide oligomerization domain (NOD)-like receptor protein (NLRP3) inflammasome is reported to be widely involved with the pathology of central nervous system disorders. But the role of NLRP3 in diffuse axonal injury (DAI) are rarely reported. The purpose of this study was to investigate the expression of NLRP3 after diffuse axonal injury and the role of NLRP3 in axonal injures. The lateral head rotation device was used to establish DAI model of rats. Immunohistochemical staining for β-amyloid precursor protein and Bielschowsky silver staining were used to assess axonal injures and axonal loss. Terminal Deoxynucleotidyl Transferase-Mediated Digoxigenin-dUTP-Biotin Nick-End Labelling Assay was used to detect cell apoptosis. Brain water content was used to assess cerebral edema and the modified Neurologic Severity Score was used to assess the neurological deficits. Components of NLRP3 inflammasome, such as NLRP3, apoptosis-associated speck-like (ASC) adapter protein and caspase-1, and pro-inflammatory cytokines, for example IL-18 and IL-1β, were over-expressed in early stages of DAI. MCC950, a selective small-molecule inhibitor of NLRP3 inflammasome, inhibited the over-expression of NLRP3 inflammasome and pro-inflammatory cytokines after DAI. MCC950 alleviated axonal injures and cell apoptosis. MCC950 also decreased brain water content and alleviated neurologic deficits 1day and 3days after DAI but not 7days after DAI. These results suggest that MCC950 treatment in the early stages of DAI has a time limiting effect in preventing from axonal injuries and neurological deficits, and that NLRP3 inflammasome plays an important role in axonal injures and may be a potential candidate for axonal injures following DAI.

Protective effect of midazolam against convulsion in neonatal rats via down-regulation of LC3 and Beclin-1 expression

Purpose: To investigate the effect of midazolam on growth of neurocytes in vitro and in neonatal rats.&#x0D; Methods: Neurocyte proliferation and activity of lactate dehydrogenase were assessed by MTT and lactate dehydrogenase assays, respectively. Western blotting was used to determine the effect of midazolam on LC3, Bax, p62 and Beclin-1 protein expressions.&#x0D; Results: The suppression of neurocyte proliferation byconvulsion was alleviated significantly (p &lt; 0.05) by midazolum treatment. Exposure of convulsion model of neurocytes to midazolum suppressed LC3, Bax, p62 and Beclin-1 protein expression. Midazolum exposure of convulsion model of neurocytes suppressed LDH, caspase-3, caspase-8 and caspase-9 activities. The 3-MA (autophagy inhibitor) treatment also significantly (p &lt; 0.05) promoted neurocyte viability after convulsion induction. In convulsion-induced neurocytes, 3-MA exposure suppressed expression of caspase-3/8/9, LC3, Bax, Beclin-1 and p62, while application of midazolum treatment to the rats with convulsion markedly decreased brain water content and neurocyte apoptosis (p &lt; 0.05). Treatment with midazolum inhibited LC3, p62 and Beclin-1 expression in the rat model of convulsion.&#x0D; Conclusion: Midazolum promotes neurocyte proliferation and inhibits edema development via downregulation of autophagy. Therefore, midazolum can potentially be used for the treatment of convulsion, but further studies need to be carried out first.&#x0D; Keywords: Convulsion, Neurocytes, Caspase, Autophagy, Mitochondrial pathway

Wnt-3a alleviates neuroinflammation after ischemic stroke by modulating the responses of microglia/macrophages and astrocytes

Neuroinflammation crucially influences functional recovery after ischemic stroke. Wnt-3a, a novel Wnt protein that specifically promotes Wnt/β-catenin signaling pathway, has been shown to regulate apoptosis and cell proliferation, but how it affects ischemic stroke-induced toxic brain inflammation remains unknown. Using a transient middle cerebral artery occlusion (tMCAO) mouse model in this study, we found that intranasal Wnt-3a-treated tMCAO mice had apparently reduced infarct volume and decreased brain water content after being allowed to recover for 72 h, as well as better neurologic outcomes on days 3, 7, and 14. Mice received Wnt-3a had significantly fewer tMCAO-induced peri-infarct TUNEL-positive cells compared with those received vehicle. Further, Wnt-3a-delivered tMCAO mice had notably fewer peri-infarct CD68-positive cells and lower ionized calcium-binding adapter molecule (Iba)-1 protein level. Wnt-3a significantly downregulated the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF)-α, and upregulated the expression of arginase 1 (Arg1) and CD206. Finally, Wnt-3a obviously decreased the number of tMCAO-induced peri-infarct glial fibrillary acidic protein (GFAP)/C3-positive cells, increased the number of GFAP/S100A10-positive cells, attenuated the protein levels of GFAP and interleukin 15 (IL15), and elevated IL33 protein level. Our findings suggest that intranasal Wnt-3a could ameliorate toxic responses of microglia/macrophages and astrocytes in ischemic brain injury, supporting that Wnt-3a might be potentially appropriate for ischemic stroke treatment functioning as an immunomodulatory agent.

Protective effect of dexmedetomidine against diabetic hyperglycemia-exacerbated cerebral ischemia/reperfusion injury: An in vivo and in vitro study

AimsDexmedetomidine (Dex) has been noted to have neuroprotective effect against cerebral ischemia-reperfusion (I/R) injury. However, the effect of Dex in diabetic hyperglycemia-exacerbated cerebral I/R injury and its underlying mechanism remain unclear. Main methodsThe infarct volume and brain edema were evaluated by 2,3,5-triphenyltetrazolium chloride staining and standard wet-dry method. Modified neurological severity score was utilized to assess the neurological deficits. The oxidative stress and inflammation were evaluated by detecting reactive oxygen species (ROS), malondialdehyde (MDA), tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay and cell count kit-8 were applied to measure cell apoptosis and viability. Key findingsDex treatment reduced infarct volume, decreased brain water content and improved neurological deficit in middle cerebral artery occlusion/reperfusion (MCAO/R) mice. Dex treatment reduced the levels of ROS, MDA, TNF-α and IL-1β in the entire middle cerebral artery territory of diabetic mice subjected to MCAO/R, as well as in primary culture of mouse hippocampal neurons stimulated with 50 mM glucose and oxygen glucose deprivation/reperfusion. Dex treatment inhibited neuronal apoptosis induced by diabetic hyperglycemia-exacerbated cerebral I/R injury. Dex upregulated nuclear factor of activated T-cells 5 (NFAT5) and Sirtuin 1 (SIRT1) expression, induced NF-E2-related factor 2 (Nrf2) translocation from cytoplasm to nucleus and inhibited the acetylation of Nrf2. However, these changes triggered by Dex treatment were abrogated by NFAT5 knockdown. SignificanceDex protects against diabetic hyperglycemia-exacerbated cerebral I/R injury through attenuation of oxidative stress, inflammation and apoptosis. The underlying mechanism is at least the NFAT5/SIRT1/Nrf2 signaling pathway dependent.

Young plasma ameliorates aging-related acute brain injury after intracerebral hemorrhage

Aging has been shown to contribute to both the declined biofunctions of aging brain and aggravation of acute brain damage, and the former could be reversed by young plasma. These results suggest that young plasma treatment may also reduce the acute brain damage induced by intracerebral hemorrhage (ICH). In the present study, we first found that the administration of young plasma significantly reduced the mortality and neurological deficit score in aging ICH rodents, which might be due to the decreased brain water content, damaged neural cells, and increased survival neurons around the perihematomal brain tissues. Then, proteomics analysis was used to screen out the potential neuroprotective circulating factors and the results showed that many factors were changed in health human plasma among young, adult, and old population. Among these significantly changed factors, the plasma insulin-like growth factor 1 (IGF-1) level was significantly decreased with age, which was further confirmed both in human and rats detected by ELISA. Additionally, the brain IGF-1 protein level in aging ICH rats was markedly decreased when compared with young rats. Interestingly, the relative decreased brain IGF-1 level was reversed by the treatment of young plasma in aging ICH rats, while the mRNA level was non-significantly changed. Furthermore, the IGF-1 administration significantly ameliorated the acute brain injury in aging ICH rats. These results indicated that young circulating factors, like IGF-1, may enter brain tissues to exert neuroprotective effects, and young plasma may be considered as a novel therapeutic approach for the clinical treatment of aging-related acute brain injury.

Recombinant milk fat globule-EGF factor-8 reduces apoptosis via integrin \u03b23/FAK/PI3K/AKT signaling pathway in rats after traumatic brain injury

Accumulating evidence suggests neuronal apoptosis has the potential to lead to more harmful effects in the pathological processes following traumatic brain injury (TBI). Previous studies have established that milk fat globule-EGF factor-8 (MFG-E8) provides neuroprotection through modulation of inflammation, oxidative stress, and especially apoptosis in cerebral ischemia and neurodegenerative disease. However, the effects of MFG-E8 on neuronal apoptosis in TBI have not yet been investigated. Therefore, we explored the role of MFG-E8 on anti-apoptosis and its potential mechanism following TBI. In the first set of experiments, adult male Sprague–Dawley (SD) rats were randomly divided into Sham and TBI groups that were each further divided into five groups representing different time points (6 h, 24 h, 72 h, and 7 days) (n = 9 each). Western blotting, quantitative real-time PCR, and immunofluorescence staining were performed to identify the expression and cellular localization of MFG-E8. In the second set of experiments, four groups were randomly assigned: Sham group, TBI + Vehicle group, and TBI + rhMFG-E8 (1 and 3 µg) (n = 15). Recombinant human MFGE8 (rhMFG-E8) was administrated as two concentrations through intracerebroventricular (i.c.v.) injection at 1 h after TBI induction. Brain water content, neurological severity score, western blotting, and immunofluorescence staining were measured at 24 and 72 h following TBI. In the final set of experiments, MFG-E8 siRNA (500 pmol/3 µl), integrin β3 siRNA (500 pmol/3 µl), and PI3K inhibitor LY294002 (5 and 20 µM) were injected i.c.v. and thereafter rats exposed to TBI. Western blotting, immunofluorescence staining, brain water content, neurological severity score, and Fluoro-Jade C (FJC) staining were used to investigate the effect of the integrin-β3/FAK/PI3K/AKT signaling pathway on MFG-E8-mediated anti-apoptosis after TBI. The expression of MFG-E8 was mainly located in microglial cells and increased to peak at 24 h after TBI. Treatment with rhMFG-E8 (3 µg) markedly decreased brain water content, improved neurological deficits, and reduced neuronal apoptosis at 24 and 72 h after TBI. rhMFG-E8 significantly enhanced the expression of integrin-β3/FAK/PI3K/AKT pathway-related components. Administration of integrin-β3 siRNA and LY294002 (5 and 20 µM) abolished the effect of rhMFG-E8 on anti-apoptosis and neuroprotection after TBI. This study demonstrated for the first time that rhMFG-E8 inhibits neuronal apoptosis and offers neuroprotection. This is suggested to occur through the modulation of the integrin-β3/FAK/PI3K/AKT signaling pathway, highlighting rhMFG-E8 as a potentially promising therapeutic strategy for TBI patients.