Cholinergic Grb2-Associated-Binding Protein 1 Regulates Cognitive Function.

Grb2-associated-binding protein 1 (Gab1) is a docking/scaffolding molecule known to play an important role in cell growth and survival. Here, we report that Gab1 is decreased in cholinergic neurons in Alzheimer's disease (AD) patients and in a mouse model of AD. In mice, selective ablation of Gab1 in cholinergic neurons in the medial septum impaired learning and memory and hippocampal long-term potentiation. Gab1 ablation also inhibited SK channels, leading to an increase in firing in septal cholinergic neurons. Gab1 overexpression, on the other hand, improved cognitive function and restored hippocampal CaMKII autorphosphorylation in AD mice. These results suggest that Gab1 plays an important role in the pathophysiology of AD and may represent a novel therapeutic target for diseases involving cholinergic dysfunction.

A switchable [2]rotaxane with two active alkenyl groups.

A novel functional [2]rotaxane containing two alkenyl bonds was designed, synthesized and characterized by 1H, 13C NMR spectroscopy and HRESI mass spectrometry. The introduction of alkenyl bonds endowed the [2]rotaxane a fascinating ability to react with versatile functional groups such as alkenyl and thiol functional groups. The reversible shuttling movement of the macrocycle between two different recognition sites on the molecular thread can be driven by external acid and base. This kind of rotaxane bearing functional groups provides a powerful platform for preparing stimuli-responsive polymers.

Visualizing peroxynitrite fluxes in endothelial cells reveals the dynamic progression of brain vascular injury.

Accumulating evidence suggests that formation of peroxynitrite (ONOO(-)) in the cerebral vasculature contributes to the progression of ischemic damage, while the underlying molecular mechanisms remain elusive. To fully understand ONOO(-) biology, efficient tools that can realize the real-time tracing of endogenous ONOO(-) fluxes are indispensable. While a few ONOO(-) fluorescent probes have been reported, direct visualization of ONOO(-) fluxes in the cerebral vasculature of live mice remains a challenge. Herein, we present a fluorescent switch-on probe (NP3) for ONOO(-) imaging. NP3 exhibits good specificity, fast response, and high sensitivity toward ONOO(-) both in vitro and in vivo. Moreover, NP3 is two-photon excitable and readily blood-brain barrier penetrable. These desired photophysical and pharmacokinetic properties endow NP3 with the capability to monitor brain vascular ONOO(-) generation after injury with excellent temporal and spatial resolution. As a proof of concept, NP3 has enabled the direct visualization of neurovascular ONOO(-) formation in ischemia progression in live mouse brain by use of two-photon laser scanning microscopy. Due to these favorable properties, NP3 holds great promise for visualizing endogenous peroxynitrite fluxes in a variety of pathophysiological progressions in vitro and in vivo.

Atg5 deficit exaggerates the lysosome formation and cathepsin B activation in mice brain after lipid nanoparticles injection.

The present study was designed to investigate the role of autophagy-lysosome signaling in the brain after application of nanoparticles. Here, lipid nanoparticles (LNs) induced elevations of Atg5, P62, LC3 and cathepsin B in mice brain. The transmission electron microscopy revealed a dramatic elevation of lysosome vacuoles colocalized with LNs cluster inside the neurons in mice brain. Immunoblot data revealed abnormal expression of cathepsin B in brain cortex following LNs injection, whereas its expression was further elevated in Atg5(+/-) mice. The importance of Atg5 in the LNs-induced autophagy-lysosome cascade was further supported by our finding that neurovascular response was exaggerated in Atg5(+/-) mice. In addition, the siRNA knockdown of Atg5 significantly blunted the increasing of LC3 and P62 in LNs-treated Neuro-2a cells. Taken together, we propose that LNs induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway. FROM THE CLINICAL EDITOR: These authors investigated autophagy-lysosome signaling in the mouse brain after application of lipid nanoparticles and report that these nanoparticles induce autophagy-lysosome signaling and neurovascular response at least partially via an Atg5-dependent pathway.

Ischemic injury promotes Keap1 nitration and disturbance of antioxidative responses in endothelial cells: a potential vasoprotective effect of melatonin.

Clinical epidemiology has indicated that the endothelial injury is a potential contributor to the pathogenesis of ischemic neurovascular damage. In this report, we assessed S-nitrosylation and nitration of Keap1 to identify downstream nitric oxide redox signaling targets into endothelial cells during ischemia. Here, oxygen-glucose deprivation (OGD) exposure initiates the nuclear import of Keap1 in endothelial cells, which interacted with nuclear-localized Nrf2, as demonstrated through co-immunoprecipitation and immunocytochemical assay. Paralleling the ischemia-induced nuclear import of Keap1, increased nitrotyrosine immunoreactivity in endothelial cells was also observed. Consistently, the addition of peroxynitrite provoked nuclear import of Keap1 and a concomitant Nrf2 nuclear import in the endothelial cells. Importantly, pharmacological inhibition of nitrosative stress by melatonin partially inhibited the OGD-induced constitutive nuclear import of Keap1 and subsequently disturbance of Nrf2/Keap1 signaling. Moreover, the effect of melatonin on nitration and S-nitrosylation of keap1 was examined in endothelial cells with 6 hr OGD exposure. Here, we demonstrated that OGD induced tyrosine nitration of Keap1, which was blocked by melatonin treatment, while there were no significant changes in S-nitrosylation of Keap1. The specific amino acid residues of Keap1 involved in tyrosine nitration were identified as Y473 by mass spectrometry. Moreover, the protective role of melatonin against damage to endothelial tight junction integrity was addressed by ZO-1 expression, paralleled with the restored heme oxygenase-1 levels during OGD. Together, our results emphasize that upon nitrosative stress, the protective effect of melatonin on endothelial cells is likely mediated at least in part by inhibition of ischemia-evoked protein nitration of Keap1, hence contributing to relieve the disturbance of Nrf2/Keap1 antioxidative signaling.

P2X7 signaling promotes microsphere embolism-triggered microglia activation by maintaining elevation of Fas ligand.

BACKGROUND: The cerebral microvascular occlusion elicits microvascular injury which mimics the different degrees of stroke severity observed in patients, but the mechanisms underlying these embolic injuries are far from understood. The Fas ligand (FasL)-Fas system has been implicated in a number of pathogenic states. Here, we examined the contribution of microglia-derived FasL to brain inflammatory injury, with a focus on the potential to suppress the FasL increase by inhibition of the P2X(7)-FasL signaling with pharmacological or genetic approaches during ischemia. METHODS: The cerebral microvascular occlusion was induced by microsphere injection in experimental animals. Morphological changes in microglial cells were studied immunohistochemically. The biochemical analyses were used to examine the intracellular changes of P2X(7)/FasL signaling. The BV-2 cells and primary microglia from mice genetically deficient in P2X(7) were used to further establish a linkage between microglia activation and FasL overproduction. RESULTS: The FasL expression was continuously elevated and was spatiotemporally related to microglia activation following microsphere embolism. Notably, P2X(7) expression concomitantly increased in microglia and presented a distribution pattern that was similar to that of FasL in ED1-positive cells at pathological process of microsphere embolism. Interestingly, FasL generation in cultured microglia cells subjected to oxygen-glucose deprivation-treated neuron-conditioned medium was prevented by the silencing of P2X(7). Furthermore, FasL induced the migration of BV-2 microglia, whereas the neutralization of FasL with a blocking antibody was highly effective in inhibiting ischemia-induced microglial mobility. Similar results were observed in primary microglia from wild-type mice or mice genetically deficient in P2X(7). Finally, the degrees of FasL overproduction and neuronal death were consistently reduced in P2X(7)(-/-) mice compared with wild-type littermates following microsphere embolism insult. CONCLUSION: FasL functions as a key component of an immunoreactive response loop by recruiting microglia to the lesion sites through a P2X(7)-dependent mechanism. The specific modulation of P2X(7)/FasL signaling and aberrant microglial activation could provide therapeutic benefits in acute and subacute phase of cerebral microembolic injury.

[Effect of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus].

OBJECTIVE: To investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus. METHODS: SD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry. RESULTS: Compared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals. CONCLUSION: The disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.

[Effect of electroacupuncture on renal vascular microcirculation in diabetic mice based on in vivo two-photon microscopy imaging].

OBJECTIVE: To investigate the protective effect of electroacupuncture (EA) at "Zusanli"(ST36)and "Weiwanxiashu"(EX-B3) on capillary function around the renal tubule and renal tubule structure in diabetic mice based on two-photon microscopy (TPM) imaging, so as to providing visualizable evidence for the regulatory effect of EA on diabetic renal vascular microcirculation. METHODS: Spontaneous type Ⅱ diabetes mellitus mice (db/db) were employed for this study. Twenty db/db mice were randomly divided into model group (n=10) and EA group (n=10), and 10 db/m mice used as the control group. EA was applied to bilateral ST36 and EX-B3 for 20 min/time, 6 times a week for 6 weeks. The body weight was recorded and the fasting blood glucose measured before and after the intervention. The urine production and water consumption of mice in each cage were recorded after EA. The renal in vivo imaging method based on TPM was established to display the morphological structure of renal tubules, and the mouse renal blood flow velocity was detected by injecting 500 kDa dextran-fluorescein into femoral vein after the intervention. RESULTS: Compared with the control group, the proportion of mice with decreased body mass in the model group was increased, accounting for 40%, while that in the control group was 0%; and fasting blood glucose, urine production and water consumption were significantly increased in the model group (P<0.001, P<0.000 1). A renal in vivo imaging method based on TPM was successfully established, which can be applied to quantitatively analyze the renal blood flow and renal tubular diameter of mice. Based on this method, the results showed that compared with the control group, the blood flow velocity of peritubular capillary in the model group was significantly decreased (P<0.000 1, P<0.001), renal tubular cells were slightly exfoliated and the diameter of renal tubular was significantly increased (P<0.000 1). Compared with the model group, EA reduced the body weight loss ratio from 40% to 0%, and significantly decreased the fasting blood glucose, urine production and water consumption (P<0.01, P<0.000 1, P<0.001), and the blood flow velocity of peritubular capillary in the EA group was significantly increased (P<0.001, P<0.05) and tubule dilatation significantly alleviated (P<0.0 1). CONCLUSION: EA at ST36 and EX-B3 can ameliorate renal vascular microcirculation disorder to relieve the renal structure damage and improve renal function in diabetes mice.

Regulation of the ischemia-induced autophagy-lysosome processes by nitrosative stress in endothelial cells.

The cellular mechanisms that underlie the diverse nitrosative stress-mediated cellular events associated with ischemic complications in endothelial cells are not yet clear. To characterize whether autophagic elements are associated with the nitrosative stress that causes endothelial damage after ischemia injury, an in vitro sustained oxygen-glucose deprivation (OGD) and an in vivo microsphere embolism model were used in the present study. Consistent with OGD-induced peroxynitrite formation, a rapid induction of microtubule-associated protein 1 light chain 3 (LC3)-I/II conversion and green fluorescent protein-LC3 puncta accumulation were observed in endothelial cells. The Western blot analyses indicated that OGD induced elevations in lysosome-associated membrane protein 2 and cathepsin B protein levels. Similar results were observed in the microvessel insult model, following occlusion of the microvessels using microsphere injections in rats. Furthermore, cultured endothelial cells treated with peroxynitrite (1-50 µm) exhibited a concentration-dependent change in the pattern of autophagy-lysosome signaling. Intriguingly, OGD-induced autophagy-lysosome processes were attenuated by PEP-19 overexpression and by a small-interfering RNA (siRNA)-mediated knockdown of eNOS. The importance of nitrosative stress in ischemia-induced autophagy-lysosome cascades is further supported by our finding that pharmacological inhibition of nitrosative stress by melatonin partially inhibits the ischemia-induced autophagy-lysosome cascade and the degradation of the tight junction proteins. Taken together, the present results demonstrate that peroxynitrite-mediated nitrosative stress at least partially potentiates autophagy-lysosome signaling during sustained ischemic insult-induced endothelial cell damage.

Melatonin ameliorates ischemic-like injury-evoked nitrosative stress: Involvement of HtrA2/PED pathways in endothelial cells.

Peroxynitrite contributes to diverse cellular stresses in the pathogenesis of ischemic complications. Here, we investigate the downstream effector signaling elements of nitrosative stress which regulate ischemia-like cell death in endothelial cells and protective effect of melatonin. When the mitochondrial membrane potential (ΔΨm) of oxygen-glucose deprivation (OGD)-treated cells was assessed using the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol -carbocyanine iodide, we observed spontaneous changes in peroxynitrite formation. Concomitantly, western blot and confocal microscopy analyses indicated that prolonged OGD exposure initiates the release of mitochondrial HtrA2 and dramatically decreases phosphoprotein enriched in astrocytes (PED or PEA-15) protein levels. Consistently, cultured endothelial cells treated with peroxynitrite (1-50 µm) exhibited a concentration-dependent release of mitochondrial HtrA2 and concomitant PED degradation in vitro. Notably, HtrA2 activation coincided with increased nitrotyrosine immunoreactivity in microvessels of rats following microsphere embolism. Additionally, the protective effect of PED overexpression in OGD-induced apoptosis was abolished by transfection with the PED(S104A/S116A) mutant. Furthermore, the effect of melatonin, an potential antioxidant, on endothelial apoptotic cascade was examined in OGD-evoked nitrosative stress. Our data showed that the application of melatonin provided significant protection against OGD-induced peroxynitrite formation and mitochondrial HtrA2 release, accompanied with a decrease in degradation PED and x-linked inhibitor of apoptosis protein, which is associated with activation of the caspase cascade. Taken together, the protective effect of melatonin is likely mediated, in part, by inhibition of peroxynitrate-mediated nitrosative stress, which in turn relieves imbalance of mitochondrial HtrA2-PED signaling and endothelial cell death.

The induction of reactive oxygen species and loss of mitochondrial Omi/HtrA2 is associated with S-nitrosoglutathione-induced apoptosis in human endothelial cells.

The pathophysiological relevance of S-nitrosoglutathione (GSNO)-induced endothelial cell injury remains unclear. The main objective of this study was to elucidate the molecular mechanisms of GSNO-induced oxidative stress in endothelial cells. Morphological evaluation through DAPI staining and propidium iodide (PI) flow cytometry was used to detect apoptosis. In cultured EA.hy926 endothelial cells, exposure to GSNO led to a time- and dose-dependent apoptotic cascade. When intracellular reactive oxygen species (ROS) production was measured in GSNO-treated cells with the fluorescent probes 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate, we observed elevated ROS levels and a concomitant loss in mitochondrial membrane potential, indicating that GSNO-induced death signaling is mediated through a ROS-mitochondrial pathway. Importantly, we found that peroxynitrite formation and Omi/HtrA2 release from mitochondria were involved in this phenomenon, whereas changes of death-receptor dependent signaling were not detected in the same context. The inhibition of NADPH oxidase activation and Omi/HtrA2 by a pharmacological approach provided significant protection against caspase-3 activation and GSNO-induced cell death, confirming that GSNO triggers the death cascade in endothelial cells in a mitochondria-dependent manner. Taken together, our results indicate that ROS overproduction and loss of mitochondrial Omi/HtrA2 play a pivotal role in reactive nitrogen species-induced cell death, and the modulation of these pathways can be of significant therapeutic benefit.

Visualizing Autophagic Flux during Endothelial Injury with a Pathway-Inspired Tandem-Reaction Based Fluorogenic Probe.

Autophagy is a dynamic and complicated catabolic process. Imaging autophagic flux can clearly advance knowledge of its pathophysiology significance. While the most common way autophagy is imaged relies on fluorescent protein-based probes, this method requires substantial genetic manipulation that severely restricts the application. Small fluorescent probes capable of tracking autophagic flux with good spatiotemporal resolution are highly demanable. Methods: In this study, we developed a small-molecule fluorogenic probe (AFG-1) that facilitates real-time imaging of autophagic flux in both intact cells and live mice. AFG-1 is inspired by the cascading nitrosative and acidic microenvironments evolving during autophagy. It operates over two sequential steps. In the first step, AFG-1 responds to the up-regulated peroxynitrite at the initiation of autophagy by its diphenylamino group being oxidatively dearylated to yield a daughter probe. In the second step, the daughter probe responds to the acidic autolysosomes at the late stage of autophagy by being protonated. Results: This pathway-dependent mechanism has been confirmed first by sequentially sensing ONOO- and acid in aqueous solution, and then by imaging autophagic flux in live cells. Furthermore, AFG-1 has been successfully applied to visualize autophagic flux in real-time in live mice following brain ischemic injury, justifying its robustness. Conclusion: Due to the specificity, easy operation, and the dynamic information yielded, AFG-1 should serve as a potential tool to explore the roles of autophagy under various pathological settings.

Myt1l induced direct reprogramming of pericytes into cholinergic neurons.

OBJECTIVE: The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. METHODS AND RESULTS: Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30 days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. CONCLUSIONS: Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

Endothelial ErbB4 deficit induces alterations in exploratory behavior and brain energy metabolism in mice.

AIMS: The receptor tyrosine kinase ErbB4 is present throughout the primate brain and has a distinct functional profile. In this study, we investigate the potential role of endothelial ErbB4 receptor signaling in the brain. RESULTS: Here, we show that the endothelial cell-specific deletion of ErbB4 induces decreased exploratory behavior in adult mice. However, the water maze task for spatial memory and the memory reconsolidation test reveal no changes; additionally, we observe no impairment in CaMKII phosphorylation in Cdh5Cre;ErbB4f/f mice, which indicates that the endothelial ErbB4 deficit leads to decreased exploratory activity rather than direct memory deficits. Furthermore, decreased brain metabolism, which was measured using micro-positron emission tomography, is observed in the Cdh5Cre;ErbB4f/f mice. Consistently, the immunoblot data demonstrate the downregulation of brain Glut1, phospho-ULK1 (Ser555), and TIGAR in the endothelial ErbB4 conditional knockout mice. Collectively, our findings suggest that endothelial ErbB4 plays a critical role in regulating brain function, at least in part, through maintaining normal brain energy homeostasis. CONCLUSIONS: Targeting ErbB4 or the modulation of endothelial ErbB4 signaling may represent a rational pharmacological approach to treat neurological disorders.

Calpain-Dependent ErbB4 Cleavage Is Involved in Brain Ischemia-Induced Neuronal Death.

Disturbance of neuregulin-1ß/ErbB4 signaling is considered to be associated with brain ischemia, but the mechanisms of this disruption are largely unknown. In the present study, we provide evidence that degradation of ErbB4 is involved in neuronal cell death in response to ischemia. Our data showed that the application of neuregulin-1ß provided significant protection against oxygen-glucose deprivation (OGD)-induced neuronal death as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide flow cytometry analysis and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining. Furthermore, neuregulin-1ß treatment significantly reduced the infarct volume of ischemic mice, and this result was not seen in the ErbB4 knockout mice. We found that brain ischemia induced the breakdown of ErbB4 in a time-dependent manner in vivo, but not that of ErbB2. In vitro studies further indicated that recombinant calpain induced the cleavage of ErbB4 in a dose-dependent way, whereas the calpain inhibitor significantly reduced the OGD-induced ErbB4 breakdown. Additionally, OGD-induced apoptosis was partially abolished by transfection with the ErbB4E872K mutant. Taken together, neuregulin-1ß elicits its neuroprotective effect in an ErbB4-dependent manner, and the cleavage of ErbB4 by calpain contributes to a neuronal cell death cascade during brain ischemia.

Functional Genomic Analyses Identify Pathways Dysregulated in Animal Model of Autism.

BACKGROUND: Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders that display complicated behavioral symptoms. METHODS: Using gene expressing profiling and the weighted gene co-expression network analysis (WGCNA), we studied genes coregulated by similar factors such as genetic variants or environmental effects in the hippocampus in an animal model of autism. RESULTS: From microarray data, we identified 21,388 robustly expressed genes of which 721 genes were found to be differently expressed in the valproic acid-treated group compared to the control group. WGCNA identified multiple co-expression modules known to associate with cognitive function, inflammation, synaptic, and positive regulation of protein kinase activating. Many of these modules, however, have not been previously linked to autism spectrum disorders which included G-protein signaling, immunity, and neuroactive ligand-receptor interaction pathway. The downregulation of the highly connected (hub) genes Taar7h and Taar7b in neuroactive ligand-receptor interaction pathway was validated by qRT-PCR. Immunoblotting and immunohistochemistry further showed that TAAR7 expression was downregulated not only in valproic acid-treated animals, but also BTBR T+tf/J mice. CONCLUSIONS: This study highlights the advantages of gene microarrays to uncover co-expression modules associated with autism and suggests that Taars and related gene regulation networks may play a significant role in autism.

Valproic Acid Influences MTNR1A Intracellular Trafficking and Signaling in a ß-Arrestin 2-Dependent Manner.

Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled ß-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a ß-arrestin 2-dependent manner.

P2RX7 sensitizes Mac-1/ICAM-1-dependent leukocyte-endothelial adhesion and promotes neurovascular injury during septic encephalopathy.

Septic encephalopathy (SE) is a critical factor determining sepsis mortality. Vascular inflammation is known to be involved in SE, but the molecular events that lead to the development of encephalopathy remain unclear. Using time-lapse in vivo two-photon laser scanning microscopy, we provide the first direct evidence that cecal ligation and puncture in septic mice induces microglial trafficking to sites adjacent to leukocyte adhesion on inflamed cerebral microvessels. Our data further demonstrate that septic injury increased the chemokine CXCL1 level in brain endothelial cells by activating endothelial P2RX7 and eventually enhanced the binding of Mac-1 (CD11b/CD18)-expressing leukocytes to endothelial ICAM-1. In turn, leukocyte adhesion upregulated endothelial CX3CL1, thereby triggering microglia trafficking to the injured site. The sepsis-induced increase in endothelial CX3CL1 was abolished in CD18 hypomorphic mutant mice. Inhibition of the P2RX7 pathway not only decreased endothelial ICAM-1 expression and leukocyte adhesion but also prevented microglia overactivation, reduced brain injury, and consequently doubled the early survival of septic mice. These results demonstrate the role of the P2RX7 pathway in linking neurovascular inflammation to brain damage in vivo and provide a rationale for targeting endothelial P2RX7 for neurovascular protection during SE.

Targeted therapy of brain ischaemia using Fas ligand antibody conjugated PEG-lipid nanoparticles.

The translation of experimental stroke research from the laboratory to successful clinical practice remains a formidable challenge. We previously reported that PEGylated-lipid nanoparticles (PLNs) effectively transport across the blood-brain barrier along with less inflammatory responses. In the present study, PLNs conjugated to Fas ligand antibody that selectively present on brain ischaemic region were used for therapeutic targeting. Fluorescent analysis of the mice brain show that encapsulated 3-n-Butylphthalide (dl-NBP) in PLNs conjugated with Fas ligand antibody effectively delivered to ipsilateral region of ischaemic brain. Furthermore, the confocal immunohistochemical study demonstrated that brain-targeted nanocontainers specifically accumulated on OX42 positive microglia cells in ischaemic region of mice model. Finally, dl-NBP encapsulated nano-drug delivery system is resulted in significant improvements in brain injury and in neurological deficit after ischaemia, with the significantly reduced dosages versus regular dl-NBP. Overall, these data suggests that PLNs conjugated to an antibody specific to the Fas ligand constituted an ideal brain targeting drug delivery system for brain ischaemia.

In vivo two-photon fluorescence microscopy reveals disturbed cerebral capillary blood flow and increased susceptibility to ischemic insults in diabetic mice.

AIMS: Diabetes mellitus increases the risk of stroke, but the mechanisms are unclear. The present study tested the hypothesis that diabetes mellitus disturbs the brain microcirculation and increases the susceptibility to cerebral damage in a middle cerebral artery occlusion (MCAO) model of ischemia. METHODS: Diabetes was induced by streptozocin in mice expressing green fluorescent protein in endothelial cells (Tie2-GFP mice). Four weeks later, they were subjected to transient (20 min) MCAO. In vivo blood flow was measured by two-photon laser-scanning microscopy (TPLSM) in cerebral arteries, veins, and capillaries. RESULTS: There was a significant decrease in red blood cell (RBC) velocity in capillaries in diabetic mice as assessed by TPLSM, yet the regional cerebral blood flow, as assessed by laser Doppler flowmetry, was maintained. Brain capillary flow developed turbulence after MCAO only in diabetic mice. These mice sustained increased neurological deficits after MCAO which were accompanied by an exaggerated degradation of tight junction proteins and blunted CaMKII phosphorylation in cerebral tissues indicating disruption of the blood-brain barrier and disturbed cognitive potential. CONCLUSION: Diabetic mice are more susceptible to disturbances of cerebral capillary blood flow which may predispose them to neurovascular defects following ischemia.