Roles of mechanosensitive ion channel PIEZO1 in the pathogenesis of brain injury after experimental intracerebral hemorrhage.

Secondary brain injury after intracerebral hemorrhage (ICH) is the main cause of poor prognosis in ICH patients, but the underlying mechanisms remain less known. The involvement of Piezo1 in brain injury after ICH was studied in a mouse model of ICH. ICH was established by injecting autologous arterial blood into the basal ganglia in mice. After vehicle, Piezo1 blocker, GsMTx4, Piezo1 activator, Yoda-1, or together with mannitol (tail vein injection) was injected into the left lateral ventricle of mouse brain, Piezo1 level and the roles of Piezo1 in neuronal injury, brain edema, and neurological dysfunctions after ICH were determined by the various indicated methods. Piezo1 protein level in neurons was significantly upregulated 24 h after ICH in vivo (human and mice). Piezo1 protein level was also dramatically upregulated in HT22 cells (a murine neuron cell line) cultured in vitro 24 h after hemin treatment as an in vitro ICH model. GsMTx4 treatment or together with mannitol significantly downregulated Piezo1 and AQP4 levels, markedly increased Bcl2 level, maintained more neurons alive, considerably restored brain blood flow, remarkably relieved brain edema, substantially decreased serum IL-6 level, and almost fully reversed the neurological dysfunctions at ICH 24 h group mice. In contrast, Yoda-1 treatment achieved the opposite effects. In conclusion, Piezo1 plays a crucial role in the pathogenesis of brain injury after ICH and may be a target for clinical treatment of ICH.

Ligation of cervical lymphatic vessels decelerates blood clearance and worsens outcomes after experimental subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is characterized by the extravasation of blood into the subarachnoid space, in which erythrocyte lysis is the primary contributor to cell death and brain injuries. New evidence has indicated that meningeal lymphatic vessels (mLVs) are essential in guiding fluid and macromolecular waste from cerebrospinal fluid (CSF) into deep cervical lymph nodes (dCLNs). However, the role of mLVs in clearing erythrocytes after SAH has not been completely elucidated. Hence, we conducted a cross-species study. Autologous blood was injected into the subarachnoid space of rabbits and rats to induce SAH. Erythrocytes in the CSF were measured with/without deep cervical lymph vessels (dCLVs) ligation. Additionally, prior to inducing SAH, we administered rats with vascular endothelial growth factor C (VEGF-C), which is essential for meningeal lymphangiogenesis and maintaining integrity and survival of lymphatic vessels. The results showed that the blood clearance rate was significantly lower after dCLVs ligation in both the rat and rabbit models. DCLVs ligation aggravated neuroinflammation, neuronal damage, brain edema, and behavioral impairment after SAH. Conversely, the treatment of VEGF-C enhanced meningeal lymphatic drainage of erythrocytes and improved outcomes in SAH. In summary, our research highlights the indispensable role of the meningeal lymphatic pathway in the clearance of blood and mediating consequences after SAH.

OXCT1 regulates hippocampal neurogenesis and alleviates cognitive impairment via the Akt/GSK-3ß/ß-catenin pathway after subarachnoid hemorrhage.

BACKGROUND: Subarachnoid hemorrhage (SAH) is a life-threatening neurological disease that usually has a poor prognosis. Neurogenesis is a potential therapeutic target for brain injury. Ketone metabolism also plays neuroprotective roles in many neurological disorders. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme of ketone body oxidation. In this study, we explored whether increasing ketone oxidation by upregulating OXCT1 in neurons could promote neurogenesis after SAH, and evaluated the potential mechanism involved in this process. METHODS: The ß-hydroxybutyrate content was measured using an enzymatic colorimetric assay. Adeno-associated virus targeting neurons was injected to overexpress OXCT1, and the expression and localization of proteins were evaluated by western blotting and immunofluorescence staining. Adult hippocampal neurogenesis was evaluated by dual staining with doublecortin and 5-Ethynyl-2'-Deoxyuridine. LY294002 was intracerebroventricularly administered to inhibit Akt activity. The Morris water maze and Y-maze tests were employed to assess cognitive function after SAH. RESULTS: The results showed that OXCT1 expression and hippocampal neurogenesis significantly decreased in the early stage of SAH. Overexpression of OXCT1 successfully increased hippocampal neurogenesis via activation of Akt/GSK-3ß/ß-catenin signaling and improved cognitive function, both of which were reversed by administration of LY294002. CONCLUSIONS: OXCT1 regulated hippocampal ketone body metabolism and increased neurogenesis through mechanisms mediated by the Akt/GSK-3ß/ß-catenin pathway, improving cognitive impairment after SAH.

Astrocyte-derived hepcidin aggravates neuronal iron accumulation after subarachnoid hemorrhage by decreasing neuronal ferroportin1.

Iron accumulation is one of the most essential pathological events after subarachnoid hemorrhage (SAH). Ferroportin1 (FPN1) is the only transmembrane protein responsible for exporting iron. Hepcidin, as the major regulator of FPN1, is responsible for its degradation. Our study investigated how the interaction between FPN1 and hepcidin contributes to iron accumulation after SAH. We found that iron accumulation aggravated after SAH, along with decreased FPN1 in neurons and increased hepcidin in astrocytes. After knocking down hepcidin in astrocytes, the neuronal FPN1 significantly elevated, thus attenuating iron accumulation. After SAH, p-Smad1/5 and Smad4 tended to translocate into the nucleus. Moreover, Smad4 combined more fragments of the promoter region of Hamp after OxyHb stimulation. By knocking down Smad1/5 or Smad4 in astrocytes, FPN1 level restored and iron overload attenuated, leading to alleviated neuronal cell death and improved neurological function. However, the protective role disappeared after recombinant hepcidin administration. Therefore, our study suggests that owing to the nuclear translocation of transcription factors p-Smad1/5 and Smad4, astrocyte-derived hepcidin increased significantly after SAH, leading to a decreased level of neuronal FPN1, aggravation of iron accumulation, and worse neurological outcome.

Aloperine targets lysosomes to inhibit late autophagy and induces cell death through apoptosis and paraptosis in glioblastoma.

Glioblastoma (GBM) is an aggressive intracranial tumour, and current chemotherapy regimens have limited efficacy. Aloperine (ALO), a natural alkaline compound, has shown potential as an antitumor agent. However, the effect of ALO against GBM remains unclear. This study aimed to investigate the function of ALO in treating GBM. U87, A172, and GL261 cell lines were used for in vitro experiments, and GL261 was also used to establish in vivo models. The results showed that ALO inhibited the proliferation of GBM cells by cell cycle arrest and apoptosis. Furthermore, autophagy was found to play a critical role, suggested by observation of autophagosomes under the transmission electron microscopy. It was discovered for the first time that ALO targeted lysosomes directly in glioma cells, tested by fluo-rescence-labelled ALO and organelle-localizing probes. In addition, ALO inhibited late autophagy and induced paraptosis in GBM, verified by classical gene expression changes in qPCR and western blotting. Also, ALO inhibited tumour growth and acted synergistically with temozolomide in intracranial glioma mice models in vivo. Our findings suggest that ALO targets lysosomes to inhibit late autophagy in GBM, inducing cell cycle arrest, paraptosis, and apoptosis. ALO may therefore be a promising therapeutic agent for the treatment of GBM.

Increased NOX2 expression in astrocytes leads to eNOS uncoupling through dihydrofolate reductase in endothelial cells after subarachnoid hemorrhage.

Introduction: Endothelial nitric oxide synthase (eNOS) uncoupling plays a significant role in acute vasoconstriction during early brain injury (EBI) after subarachnoid hemorrhage (SAH). Astrocytes in the neurovascular unit extend their foot processes around endothelia. In our study, we tested the hypothesis that increased nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) expression in astrocytes after SAH leads to eNOS uncoupling. Methods: We utilized laser speckle contrast imaging for monitoring cortical blood flow changes in mice, nitric oxide (NO) kits to measure the level of NO, and a co-culture system to study the effect of astrocytes on endothelial cells. Moreover, the protein levels were assessed by Western blot and immunofluorescence staining. We used CCK-8 to measure the viability of astrocytes and endothelial cells, and we used the H2O2 kit to measure the H2O2 released from astrocytes. We used GSK2795039 as an inhibitor of NOX2, whereas lentivirus and adeno-associated virus were used for dihydrofolate reductase (DHFR) knockdown in vivo and in vitro. Results: The expression of NOX2 and the release of H2O2 in astrocytes are increased, which was accompanied by a decrease in endothelial DHFR 12 h after SAH. Moreover, the eNOS monomer/dimer ratio increased, leading to a decrease in NO and acute cerebral ischemia. All of the above were significantly alleviated after the administration of GSK2795039. However, after knocking down DHFR both in vivo and in vitro, the protective effect of GSK2795039 was greatly reversed. Discussion: The increased level of NOX2 in astrocytes contributes to decreased DHFR in endothelial cells, thus aggravating eNOS uncoupling, which is an essential mechanism underlying acute vasoconstriction after SAH.

Neuroprotective mechanisms of OXCT1 via the SIRT3-SOD2 pathway after traumatic brain injury.

BACKGROUND: Ketones are not only utilized to produce energy but also play a neuroprotective role in many neurodegenerative diseases. However, whether this process has an impact on secondary brain damage after traumatic brain injury (TBI) remains unknown. OXCT1 (3-Oxoacid CoA-Transferase 1) is the rate-limiting enzyme in the intra-neuronal utilization of ketones. In this study, we investigated whether reduced expression of OXCT1 after TBI could impact neuroprotective mechanisms and exacerbate neurological dysfunction. MATERIALS AND METHODS: Experimental TBI was induced by a modified version of the weight drop model, it is a model of severe head trauma. Expression of OXCT1 in the injured hippocampus of mice was measured at different time points using immunoblotting assays. The release of abnormal mitochondrial cytochrome c from neurons of the mouse injured lateral hippocampus was measured 1 week after TBI using immunoblotting assays. Neuronal death was assessed by Nissl staining and the level of reactive oxygen species (ROS) within the neurons of the injured lateral hippocampus was assessed by Dihydroethidium staining. RESULTS: OXCT1 was overexpressed in hippocampal neurons by injection of adeno-associated virus into the lateral ventricle. OXCT1 expression levels decreased significantly 1 week post-TBI. After comparing the data obtained from different groups of mice, OXCT1 was found to significantly increase the expression of SIRT3 and reduce the proportion of acetylated SOD2, thus decreasing the production of ROS in the injured hippocampal neurons, reducing neuronal death, and improving cognitive function. CONCLUSIONS: OXCT1 has a critical previously unappreciated protective role in neurological impairment following TBI via the SIR3-SOD2 pathway. These findings highlight the potential of OXCT1 as a simple treatment for patients with TBI.

Endothelial NOX4 aggravates eNOS uncoupling by decreasing dihydrofolate reductase after subarachnoid hemorrhage.

Endothelial malfunction is a major contributor to early or delayed vasospasm after subarachnoid hemorrhage (SAH). As a representative form of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) uncoupling leads to a reduction in nitric oxide (NO) generated by endothelial cells. In this study, we investigated how the interaction between endothelial NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4) and DHFR (dihydrofolate reductase) contributes to eNOS uncoupling after SAH. Setanaxib and the adeno-associated virus (AAV) targeting brain vascular endothelia were injected through the tail vein and the expression and localization of proteins were examined by western blot and immunofluorescence staining. The NO content was measured using the NO assay kit, and laser speckle contrast imaging was used to assess cortical perfusion. ROS (reactive oxygen species) level was detected by DHE (dihydroethidium) staining, DCFH-DA (2',7'-dichlorofluorescin diacetate) staining and H2O2 (hydrogen peroxide) measurement. The Garcia score was employed to examine neurological function. Setanaxib is widely used for its preferential inhibition for NOX1/4 over other NOX isoforms. After endothelial NOX4 was inhibited by Setanaxib in a mouse model of SAH, the endothelial DHFR level was significantly elevated, which attenuated eNOS uncoupling, increased cortical perfusion, and improved the neurological function. The protective role of inhibiting endothelial NOX4, however, disappeared after knocking down endothelial DHFR. Our results suggest that endothelial DHFR decreased significantly because of the elevated level of endothelial NOX4, which aggravated eNOS uncoupling after SAH, leading to decreased cortical perfusion and worse neurological outcome.

Expression and potential role of FOSB in glioma.

Background: FOSB is reported to be an oncogene in a variety of tumors. However, the expression and role of FOSB in glioma remain obscure. In this study, we aimed to explore the expression of FOSB in glioma and its biological role in glioblastoma multiforme (GBM). Methods: Western blot, immunohistochemical staining, and quantitative real-time polymerase chain reaction (RT-qPCR) were used to detect the expression of FOSB in clinical samples. FOSB was knocked down in cells to determine the effects of FOSB on the phenotypic changes of tumors by plate cloning, CCK-8 assay, and Transwell assay. Finally, subcutaneous tumorigenesis in nude mice was used to observe the tumorigenesis of glioma cell lines after the knockdown of the FOSB gene. Results: FOSB expression was higher in glioma compared with normal brain tissue. After the downregulation of FOSB, the expression of cleaved caspase-3 increased. Plate cloning and CCK-8 experiments showed that the proliferation of glioma cell lines decreased. The Transwell assay demonstrated that the glioblastoma cell lines had lower migration ability after the knockdown of FOSB. Finally, the tumor volume of U87 glioma cells in group sh-FOSB was smaller than that in the control group. The TUNEL staining in vitro showed that the apoptosis of sh-FOSB glioma cells increased. Conclusion: FOSB was highly expressed in glioma tissues. The viability of glioma cells decreased, and the ability of glioma cells to proliferate and migrate was reduced when FOSB was downregulated. Hence, FOSB may promote the development and migration of gliomas.

Activation of HIF-1α/VEGF-A pathway by deferoxamine ameliorates retinal hypoxia in a rat subarachnoid hemorrhage model.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is associated with sustained vasoconstriction in retinal vessels and vasoconstriction leads to retinal ischemia and hypoxia. Our previous finding also revealed the changes in hypoxia-related elements in the retina after SAH, further lending weight to the hypothesis that retinal vasospasm and hypoxia after SAH. Deferoxamine is a high-affinity iron chelator with reported neuroprotective effects against stroke. Here, we aimed to explore the effects of deferoxamine on retinal hypoxia after SAH. METHODS: SAH was established and deferoxamine was injected intraperitoneally for 3 days in the treatment group. To detect retinal new vessels, platelet endothelial cell adhesion molecule (CD31) was labeled by immunofluorescence and immunohistochemistry. Furthermore, the effects of deferoxamine on the expression of vascular endothelial growth factor A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) were revealed by western blot analysis. RESULTS: The immunofluorescence and immunohistochemical staining of CD31 revealed a marked increase in new vessels in the retinal ganglion cell layer after deferoxamine treatment. By western blot analysis, HIF-1α and VEGF-A increased gradually in the first day and then rebounded to a new level on day 7. A deferoxamine-induced increase in HIF-1α/VEGF-A expression was also confirmed by western blot. CONCLUSIONS: Our findings suggest that modulating the application of deferoxamine may offer therapeutic approaches to alleviate retinal complications after SAH.

Peroxisome proliferator-activated receptor-γ ameliorates neuronal ferroptosis after traumatic brain injury in mice by inhibiting cyclooxygenase-2.

Among the multiple kinds of neuronal cell death triggered by traumatic brain injury (TBI), ferroptosis, an iron-dependent lipid peroxidative regulatory cell death, has a critical role. Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear transcription factor that regulates lipid metabolism and suppresses neuronal inflammation. However, the role of PPARγ in neuronal ferroptosis induced by TBI remains unclear. Here, we investigated the regulatory effect of PPARγ on neuronal ferroptosis in a weight-drop TBI model in vivo and an RAS-selective lethal 3 (RSL3)-activated ferroptotic neuronal model in vitro. PPARγ was mainly localized in the nucleus of neurons and was decreased in both the in vivo TBI model and the in vitro ferroptotic neuronal model. The addition of a specific agonist, pioglitazone, activated PPARγ, which protected neuronal function post-TBI in vivo and increased the viability of ferroptotic neurons in vitro. Further investigation suggested that PPARγ probably attenuates neuronal ferroptosis by downregulating cyclooxygenase-2 (COX2) protein expression levels in vivo and in vitro. This study revealed the relationship among PPARγ, ferroptosis and TBI and identified a potential target for comprehensive TBI treatment.

Inhibition of TRPA1 Attenuates Oxidative Stress-induced Damage After Traumatic Brain Injury via the ERK/AKT Signaling Pathway.

Neuron apoptosis is a feature of secondary injury after traumatic brain injury (TBI). Evidence implies that excess calcium (Ca2+) ions and reactive oxidative species (ROS) play critical roles in apoptosis. In reaction to increased ROS, the anti-oxidative master transcription factor, Transient receptor potential Ankyrin 1 (TRPA1) allows Ca2+ ions to enter cells. However, the effect of TBI on the expression of TRPA1 and the role of TRPA1 in TBI are unclear. In the present study, TBI in the mouse brain was simulated using the weight-drop model. The process of neuronal oxidative stress was simulated in HT22 neuronal cells by treatment with hydrogen peroxide. We found that TRPA1 was significantly upregulated in neurons at 24 h after TBI. Neuronal apoptosis was increased in the in vivo and in vitro models; however, this increase was reduced by the functional inhibition of TRPA1 in both models. After TBI, TRPA1 was upregulated via nuclear factor, erythroid 2 like 2 (Nrf2) in neurons. TRPA1-mediated neuronal apoptosis after TBI might be achieved in part through the CaMKII/AKT/ERK signaling pathway. To sum up, TBI-triggered TRPA1 upregulation in neurons is mediated by Nrf2 and the functional blockade of TRPA1 attenuates neuronal apoptosis and improves neuronal dysfunction, partially mediated through the activation of the calcium/calmodulin dependent protein kinase II (CaMKII) extracellular regulated kinase (ERK)/protein kinase B (AKT) signaling pathway. Our results suggest that functional blockade of TRPA1 might be a promising therapeutic intervention related to ROS and Nrf2 in TBI.

Restoration of Brain Angiotensin-Converting Enzyme 2 Alleviates Neurological Deficits after Severe Traumatic Brain Injury via Mitigation of Pyroptosis and Apoptosis.

Clinically, the renin-angiotensin-aldosterone system is activated intensely in patients with moderate to severe traumatic brain injury (TBI). Increased angiotensin II in circulatory blood after TBI can enter the brain through the disrupted blood-brain barrier. Angiotensin-converting enzyme 2 (ACE2) is an enzyme that metabolizes angiotensin II into angiotensin (1-7), which has been shown to have neuroprotective results. The expression and role of ACE2 in the brain after TBI remains elusive, however. We found that ACE2 protein abundance was downregulated around the contusional area in the brains of both humans and mice. Endogenous ACE2 was expressed in neurons, astrocytes, and microglia in the cortex of the mouse brain. Administration of recombinant human ACE2 intracerebroventricularly alleviated neurological defects after TBI in mice. Treatment of recombinant human ACE2 suppressed TBI-induced increase of angiotensin II and the decrease of angiotensin (1-7) in the brain, mitigated neural cell death, reduced the activation of NLRP3 and caspase3, decreased phosphorylation of mitogen-activated protein kinases, and nuclear factor kappa B, and reduced inflammatory cytokines tumor necrosis factor alpha and interleukin-1ß. Administration of ACE2 enzyme activator diminazene aceturate intraperitoneally rescued downregulation of ACE2 enzymatic activity and protein abundance in the brain. Diminazene aceturate treatment once per day in the acute stage after TBI alleviated long-term cognitive defects and neuronal loss in mice. Collectively, these results indicated that restoration of ACE2 alleviated neurological deficits after TBI by mitigation of pyroptosis and apoptosis.

Dobutamine promotes the clearance of erythrocytes from the brain to cervical lymph nodes after subarachnoid hemorrhage in mice.

Background: Erythrocytes and their breakdown products in the subarachnoid space (SAS) are the main contributors to the pathogenesis of subarachnoid hemorrhage (SAH). Dobutamine is a potent ß1-adrenoreceptor agonist that can increase cardiac output, thus improving blood perfusion and arterial pulsation in the brain. In this study, we investigated whether the administration of dobutamine promoted the clearance of red blood cells (RBCs) and their degraded products via meningeal lymphatic vessels (mLVs), thus alleviating neurological deficits in the early stage post-SAH. Materials and methods: Experimental SAH was induced by injecting autologous arterial blood into the prechiasmatic cistern in male C57BL/6 mice. Evans blue was injected into the cisterna magna, and dobutamine was administered by inserting a femoral venous catheter. RBCs in the deep cervical lymphatic nodes (dCLNs) were evaluated by hematoxylin-eosin staining, and the hemoglobin content in dCLNs was detected by Drabkin's reagent. The accumulation of RBCs in the dura mater was examined by immunofluorescence staining, neuronal death was evaluated by Nissl staining, and apoptotic cell death was evaluated by TUNEL staining. The Morris water maze test was used to examine the cognitive function of mice after SAH. Results: RBCs appeared in dCLNs as early as 3 h post-SAH, and the hemoglobin in dCLNs peaked at 12 h after SAH. Dobutamine significantly promoted cerebrospinal fluid (CSF) drainage from the SAS to dCLNs and obviously reduced the RBC residue in mLVs, leading to a decrease in neuronal death and an improvement in cognitive function after SAH. Conclusion: Dobutamine administration significantly promoted RBC drainage from cerebrospinal fluid in the SAS via mLVs into dCLNs, ultimately relieving neuronal death and improving cognitive function.

A20 Establishes Negative Feedback With TRAF6/NF-κB and Attenuates Early Brain Injury After Experimental Subarachnoid Hemorrhage.

Nuclear factor (NF)-κB-ty -50mediated neuroinflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). As an important negative feedback regulator of NF-κB, A20 is essential for inflammatory homeostasis. Herein, we tested the hypothesis that A20 attenuates EBI by establishing NF-κB-associated negative feedback after experimental SAH. In vivo and in vitro models of SAH were established. TPCA-1 and lentivirus were used for NF-κB inhibition and A20 silencing/overexpression, respectively. Cellular localization of A20 in the brain was determined via immunofluorescence. Western blotting and enzyme-linked immunosorbent assays were applied to observe the expression of members of the A20/tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB pathway and inflammatory cytokines (IL-6, IL-1ß, TNF-α). Evans blue staining, TUNEL staining, Nissl staining, brain water content, and modified Garcia score were performed to evaluate the neuroprotective effect of A20. A20 expression by astrocytes, microglia, and neurons was increased at 24 h after SAH. A20 and inflammatory cytokine levels were decreased while TRAF6 expression was elevated after NF-κB inhibition. TRAF6, NF-κB, and inflammatory cytokine levels were increased after A20 silencing but suppressed with A20 overexpression. Also, Bcl-2, Bax, MMP-9, ZO-1 protein levels; Evans blue, TUNEL, and Nissl staining; brain water content; and modified Garcia score showed that A20 exerted a neuroprotective effect after SAH. A20 expression was regulated by NF-κB. In turn, increased A20 expression inhibited TRAF6 and NF-κB to reduce the subsequent inflammatory response. Our data also suggest that negative feedback regulation mechanism of the A20/TRAF6/NF-κB pathway and the neuroprotective role of A20 to attenuate EBI after SAH.

Expression of FOXO transcription factors in the brain following traumatic brain injury.

Traumatic brain injury (TBI) is a substantial clinical and social problem worldwide, causing high morbidity and mortality along with significant economic and medical costs. Forkhead box O transcription factors (FOXOs) have been found to play a critical role in the regulation of cell functions, such as nutrient metabolism, programmed cell death, and tumor suppression. In the central nervous system, FOXOs are reported to be pivotal regulators of learning and memory, neurite outgrowth, and axonal degeneration. However, the role of FOXOs in TBI is still unknown. Here, we investigate changes in the expression of FOXOs in the acute stage following TBI. First, we evaluated the expression of FOXO proteins in the brains of humans after TBI. A TBI model was then established in mice, and the ipsilateral cerebral cortex was collected at 3 h, 6 h, 9 h, 12 h, 24 h, and 72 h post-TBI. The dynamic expression of Foxo proteins was observed. Neuron-specific localization of Foxos was detected by double immunofluorescence staining. Following TBI, FOXO proteins in the brains of humans were significantly increased. In mice, Foxo protein levels generally peaked at 24 h. By examining co-localization with neurons, the proportion of Foxo(+) neurons was found to increase following TBI and peak at 24 h. This study reveals the time-dependent and neuron-specific expression of Foxos following TBI in mice, providing insight to enhance understanding of the role of Foxos in TBI.

Deferoxamine reduces amyloid-beta peptides genesis and alleviates neural apoptosis after traumatic brain injury.

Traumatic brain injury (TBI) is recognized as the most influential risk factor for neurodegenerative diseases later in life, including Alzheimer's disease. The aberrant genesis of amyloid-ß peptides, which is triggered by TBI, is associated with the development of Alzheimer's disease. Evidence suggests that iron plays a role in both the production of amyloid-ß and its neurotoxicity, and iron overload has been noted in the brain after TBI. We therefore investigated the effects of an iron-chelating treatment on amyloid-ß genesis in a weight-drop model of TBI in mice. Human brain samples were obtained from patients undergoing surgery for severe brain trauma. The Institute of Cancer Research mice were treated with deferoxamine by intraperitoneal injection after TBI induction. Changes in amyloid-ß(1-42) were assessed using western blot and immunohistochemical staining. Ferritin was also detected using western blot to investigate iron deposition in the mice brain. Immunofluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was also performed to evaluate neural apoptosis. The amyloid-ß(1-42) was markedly elevated after TBI in both humans and mice. Deferoxamine treatment in mice significantly decreased the levels of both amyloid-ß(1-42) and ferritin in the brain, and reduced TBI-induced neural cell apoptosis. The iron chelator deferoxamine can alleviate the increase of amyloid-ß(1-42) in the brain after TBI, and may therefore be a potential therapeutic strategy to prevent TBI patients from undergoing neurodegenerative processes.

FTY720 Reduces Endothelial Cell Apoptosis and Remodels Neurovascular Unit after Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. A sequence of pathological processes occurred when there is TBI. Previous studies showed that sphingosine-1-phosphate receptor 1 (S1PR1) played a critical role in inflammatory response in the brain after TBI. Thus, the present study was designed to evaluate the effects of the S1PR1 modulator FTY720 on neurovascular unit (NVU) after experimental TBI in mice. The weight-drop TBI method was used to induce TBI. Western blot (WB) was performed to determine the levels of SIPR1, claudin-5 and occludin at different time points. FTY720 was intraperitoneally administered to mice after TBI was induced. The terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay was used to assess endothelial cell apoptosis. Immunofluorescence and WB were performed to measure the expression of tight junction proteins: claudin-5 and occludin. Evans blue (EB) permeability assay and brain water content were applied to evaluate the blood-brain barrier (BBB) permeability and brain edema. Immunohistochemistry was performed to assess the activation of astrocytes and microglia. The results showed that FTY720 administration reduced endothelial cell apoptosis and improved BBB permeability. FTY720 also attenuated astrocytes and microglia activation. Furthermore, treatment with FTY720 not only improved neurological function, but also increased the survival rate of mice significantly. These findings suggest that FTY720 administration restored the structure of the NVU after experimental TBI by decreasing endothelial cell apoptosis and attenuating the activation of astrocytes. Moreover, FTY720 might reduce inflammation in the brain by reducing the activation of microglia in TBI mice.

Activation of silent information regulator 1 exerts a neuroprotective effect after intracerebral hemorrhage by deacetylating NF-κB/p65.

Nuclear factor (NF)-κB-mediated neuroinflammation is an important mechanism of intracerebral hemorrhage (ICH)-induced neurotoxicity. Silent information regulator 1 (SIRT1) plays a multi-protective effect in a variety of diseases by deacetylating and inhibiting NF-κB/p65. However, the role of SIRT1 in brain damage following ICH remains unclear. We hypothesized that SIRT1 can protect against ICH-induced brain damage by inhibiting neuroinflammation through deacetylating NF-κB/p65. The ICH model was induced in vivo (with collagenase) and in vitro (with hemoglobin). Resveratrol and Ex527 were administered to activate or inhibit SIRT1, respectively. Western blot, immunohistochemistry, and immunofluorescence assays were performed to detect the expression of SIRT1 and p65. Enzyme-linked immunosorbent assays (ELISAs) were used to explore tumor necrosis factor (TNF)-α and interleukin (IL)-1ß release. The neurological score, brain water content, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and brain hemoglobin content were determined to evaluate the neuroprotective effect of SIRT1. SIRT1 expression was decreased, whereas the level of acetylated p65 (Ac-p65) was elevated after ICH in vivo. Moreover, hemoglobin treatment decreased the expression of SIRT1 in vitro. Activation of SIRT1 by resveratrol had a neuroprotective effect, along with decreased levels of Ac-p65, IL-1ß, TNF-α, and apoptosis after ICH. The effect of resveratrol was abolished by the SIRT1 inhibitor Ex527. Our results are consistent with the hypothesis that SIRT1 exerts a neuroprotective effect after ICH by deacetylating p65 to inhibit the NF-κB-dependent inflammatory response.