Xiaoning Han

The Transcriptome and Metabolic Gene Signature of Protoplasmic Astrocytes in the Adult Murine Cortex

Protoplasmic astrocytes are critically important to energy metabolism in the CNS. Our current understanding of the metabolic interactions between neurons and glia is based on studies using cultured cells, from which mainly inferential conclusions have been drawn as to the relative roles of neurons and glia in brain metabolism. In this study, we used functional genomics to establish the relative compartmentalization of neuronal and astrocytic metabolic pathways in the adult brain. To this end, fluorescence-activated cell sorting was used to directly isolate neurons and protoplasmic astrocytes from the cortex of adult mice. Microarray analysis showed that astrocytes and neurons each express transcripts predicting individual self-sufficiency in both glycolysis and oxidative metabolism. Surprisingly, most enzymes in the tricarboxylic acid (TCA) cycle were expressed at higher relative levels in astrocytes than in neurons. Mass spectrometric analysis of the TCA cycle intermediates confirmed that freshly isolated adult astrocytes maintained an active TCA cycle, whereas immuno-electron microscopy revealed that fine astrocytic processes encompassing synapses contained a higher density of mitochondria than surrounding cells. These observations indicate that astrocytes exhibit robust oxidative metabolism in the intact adult brain and suggest a prominent contribution of astrocytic metabolism to functional brain imaging, including BOLD (blood-oxygen level-dependent) functional magnetic resonance imaging signals.

Systemic administration of an antagonist of the ATP-sensitive receptor P2X7 improves recovery after spinal cord injury

Traumatic spinal cord injury is characterized by an immediate, irreversible loss of tissue at the lesion site, as well as a secondary expansion of tissue damage over time. Although secondary injury should, in principle, be preventable, no effective treatment options currently exist for patients with acute spinal cord injury (SCI). Excessive release of ATP by the traumatized tissue, followed by activation of high-affinity P2X7 receptors, has previously been implicated in secondary injury, but no clinically relevant strategy by which to antagonize P2X7 receptors has yet, to the best of our knowledge, been reported. Here we have tested the neuroprotective effects of a systemically administered P2X7R antagonist, Brilliant blue G (BBG), in a weight-drop model of thoracic SCI in rats. Administration of BBG 15 min after injury reduced spinal cord anatomic damage and improved motor recovery without evident toxicity. Moreover, BBG treatment directly reduced local activation of astrocytes and microglia, as well as neutrophil infiltration. These observations suggest that BBG not only protected spinal cord neurons from purinergic excitotoxicity, but also reduced local inflammatory responses. Importantly, BBG is a derivative of a commonly used blue food color (FD&C blue No. 1), which crosses the blood–brain barrier. Systemic administration of BBG may thus comprise a readily feasible approach by which to treat traumatic SCI in humans.

A Central Role of Connexin 43 in Hypoxic Preconditioning

Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.

Toxic role of prostaglandin E2 receptor EP1 after intracerebral hemorrhage in mice

Inflammatory mechanisms mediated by prostaglandins may contribute to the progression of intracerebral hemorrhage (ICH)-induced brain injury, but they are not fully understood. In this study, we examined the effect of prostaglandin E2 receptor EP1 (EP1R) activation and inhibition on brain injury in mouse models of ICH and investigated the underlying mechanism of action. ICH was induced by injecting collagenase, autologous blood, or thrombin into the striatum of middle-aged male and female mice and aged male mice. Effects of selective EP1R agonist ONO-DI-004, antagonist SC51089, and nonspecific Src family kinase inhibitor PP2 were evaluated by a combination of histologic, magnetic resonance imaging (MRI), immunofluorescence, molecular, cellular, and behavioral assessments. EP1R was expressed primarily in neurons and axons but not in astrocytes or microglia after ICH induced by collagenase. In middle-aged male mice subjected to collagenase-induced ICH, EP1R inhibition mitigated brain injury, brain edema, cell death, neuronal degeneration, neuroinflammation, and neurobehavioral deficits, whereas its activation exacerbated these outcomes. EP1R inhibition also was protective in middle-aged female mice and aged male mice after collagenase-induced ICH and in middle-aged male mice after blood- or thrombin-induced ICH. EP1R inhibition also reduced oxidative stress, white matter injury, and brain atrophy and improved functional outcomes. Histologic results were confirmed by MRI. Src kinase phosphorylation and matrix metalloproteinase-9 activity were increased by EP1R activation and decreased by EP1R inhibition. EP1R regulated matrix metalloproteinase-9 activity through Src kinase signaling, which mediated EP1R toxicity after collagenase-induced ICH. We conclude that prostaglandin E2 EP1R activation plays a toxic role after ICH through mechanisms that involve the Src kinases and the matrix metalloproteinase-9 signaling pathway. EP1R inhibition could be a novel therapeutic strategy to improve outcomes after ICH.

Cerebroprotection of Flavanol (−)-Epicatechin after Traumatic Brain Injury via Nrf2-dependent and –independent Pathways

Traumatic brain injury (TBI), which leads to disability, dysfunction, and even death, is a prominent health problem worldwide with no effective treatment. A brain-permeable flavonoid named (-)-epicatechin (EC) modulates redox/oxidative stress and has been shown to be beneficial for vascular and cognitive function in humans and for ischemic and hemorrhagic stroke in rodents. Here we examined whether EC is able to protect the brain against TBI-induced brain injury in mice and if so, whether it exerts neuroprotection by modulating the NF-E2-related factor (Nrf2) pathway. We used the controlled cortical impact model to mimic TBI. EC was administered orally at 3h after TBI and then every 24h for either 3 or 7 days. We evaluated lesion volume, brain edema, white matter injury, neurologic deficits, cognitive performance and emotion-like behaviors, neutrophil infiltration, reactive oxygen species (ROS), and a variety of injury-related protein markers. Nrf2 knockout mice were used to determine the role of the Nrf2 signaling pathway after EC treatment. In wild-type mice, EC significantly reduced lesion volume, edema, and cell death and improved neurologic function on days 3 and 28; cognitive performance and depression-like behaviors were also improved with EC administration. In addition, EC reduced white matter injury, heme oxygenase-1 expression, and ferric iron deposition after TBI. These changes were accompanied by attenuation of neutrophil infiltration and oxidative insults, reduced activity of matrix metalloproteinase 9, decreased Keap 1 expression, increased Nrf2 nuclear accumulation, and increased expression of superoxide dismutase 1 and quinone 1. However, EC did not significantly reduce lesion volume or improve neurologic deficits in Nrf2 knockout mice after TBI. Our results show that EC protects the TBI brain by activating the Nrf2 pathway, inhibiting heme oxygenase-1 protein expression, and reducing iron deposition. The latter two effects could represent an Nrf2-independent mechanism in this model of TBI.

Inhibition of neuronal ferroptosis protects hemorrhagic brain

Intracerebral hemorrhage (ICH) causes high mortality and morbidity, but our knowledge of post-ICH neuronal death and related mechanisms is limited. In this study, we first demonstrated that ferroptosis, a newly identified form of cell death, occurs in the collagenase-induced ICH model in mice. We found that administration of ferrostatin-1, a specific inhibitor of ferroptosis, prevented neuronal death and reduced iron deposition induced by hemoglobin in organotypic hippocampal slice cultures (OHSCs). Mice treated with ferrostatin-1 after ICH exhibited marked brain protection and improved neurologic function. Additionally, we found that ferrostatin-1 reduced lipid reactive oxygen species production and attenuated the increased expression level of PTGS2 and its gene product cyclooxygenase-2 ex vivo and in vivo. Moreover, ferrostatin-1 in combination with other inhibitors that target different forms of cell death prevented hemoglobin-induced cell death in OHSCs and human induced pluripotent stem cell-derived neurons better than any inhibitor alone. These results indicate that ferroptosis contributes to neuronal death after ICH, that administration of ferrostatin-1 protects hemorrhagic brain, and that cyclooxygenase-2 could be a biomarker of ferroptosis. The insights gained from this study will advance our knowledge of the post-ICH cell death cascade and be essential for future preclinical studies.

Inhibition of prostaglandin E2 receptor EP3 mitigates thrombin-induced brain injury:

Prostaglandin E2 EP3 receptor is the only prostaglandin E2 receptor that couples to multiple G-proteins, but its role in thrombin-induced brain injury is unclear. In the present study, we exposed mouse hippocampal slice cultures to thrombin in vitro and injected mice with intrastriatal thrombin in vivo to investigate the role of EP3 receptor in thrombin-induced brain injury and explore its underlying cellular and molecular mechanisms. In vitro, EP3 receptor inhibition reduced thrombin-induced hippocampal CA1 cell death. In vivo, EP3 receptor was expressed in astrocytes and microglia in the perilesional region. EP3 receptor inhibition reduced lesion volume, neurologic deficit, cell death, matrix metalloproteinase-9 activity, neutrophil infiltration, and the number of CD68+ microglia, but increased the number of Ym-1+ M2 microglia. RhoA-Rho kinase levels were increased after thrombin injection and were decreased by EP3 receptor inhibition. In mice that received an intrastriatal injection of autologous arterial blood, inhibition of thrombin activity with hirudin decreased RhoA expression compared with that in vehicle-treated mice. However, EP3 receptor activation reversed this effect of hirudin. These findings show that prostaglandin E2 EP3 receptor contributes to thrombin-induced brain damage via Rho-Rho kinase–mediated cytotoxicity and proinflammatory responses.

Pinocembrin protects hemorrhagic brain primarily by inhibiting toll-like receptor 4 and reducing M1 phenotype microglia

Neuroinflammation is a major contributor to intracerebral hemorrhage (ICH) progression, but no drug is currently available to reduce this response and protect against ICH-induced injury. Recently, the natural product pinocembrin has been shown to ameliorate neuroinflammation and is undergoing a phase II clinical trial for ischemic stroke treatment. In this study, we examined the efficacy of pinocembrin in an ICH model, and further examined its effect on microglial activation and polarization. In vivo, pinocembrin dose-dependently reduced lesion volume by ∼47.5% and reduced neurologic deficits of mice at 72h after collagenase-induced ICH. The optimal dose of pinocembrin (5mg/kg) suppressed microglial activation as evidenced by decreases in CD68-positive microglia and reduced proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Pinocembrin also reduced the number of classically activated M1-like microglia without affecting M2-like microglia in the perilesional region. Additionally, pinocembrin decreased the expression of toll-like receptor (TLR)4 and its downstream target proteins TRIF and MyD88. The protection by pinocembrin was lost in microglia-depleted mice and in TLR4lps-del mice, and pinocembrin failed to decrease the number of M1-like microglia in TLR4lps-del mice. In lipopolysaccharide-stimulated BV-2 cells or primary microglia, pinocembrin decreased M1-related cytokines and markers (IL-1β, IL-6, TNF-α, and iNOS), NF-κB activation, and TLR4 expression, but it did not interfere with TLR4/MyD88 and TLR4/TRIF interactions or affect microglial phagocytosis of red blood cells. Inhibition of the TLR4 signaling pathway and reduction in M1-like microglial polarization might be the major mechanism by which pinocembrin protects hemorrhagic brain. With anti-inflammatory properties, pinocembrin could be a promising new drug candidate for treating ICH and other acute brain injuries.

Multimodality MRI assessment of grey and white matter injury and blood-brain barrier disruption after intracerebral haemorrhage in mice

In this study, we examined injury progression after intracerebral haemorrhage (ICH) induced by collagenase in mice using a preclinical 11.7 Tesla MRI system. On T2-weighted MRI, lesion and striatal volumes were increased on day 3 and then decreased from days 7 to 28. On day 3, with an increase in striatal water content, vasogenic oedema in the perihaematomal region presented as increased T2 and increased apparent diffusion coefficient (ADC) signal. With a synchronous change in T2 and ADC signals, microglial activation peaked on day 3 in the same region and decreased over time. Iron deposition appeared on day 3 around the haematoma border but did not change synchronously with ADC signals. Vascular permeability measured by Evans blue extravasation on days 1, 3, and 7 correlated with the T1-gadolinium results, both of which peaked on day 3. On diffusion tensor imaging, white matter injury was prominent in the corpus callosum and internal capsule on day 3 and then partially recovered over time. Our results indicate that the evolution of grey/white matter injury and blood-brain barrier disruption after ICH can be assessed with multimodal MRI, and that perihaematomal vasogenic oedema might be attributable to microglial activation, iron deposition, and blood-brain barrier breakdown.

Cerebroprotection by the neuronal PGE2 receptor EP2 after intracerebral hemorrhage in middle-aged mice.

Inflammatory responses mediated by prostaglandins such as PGE2 may contribute to secondary brain injury after intracerebral hemorrhage (ICH). However, the cell-specific signaling by PGE2 receptor EP2 differs depending on whether the neuropathic insult is acute or chronic. Using genetic and pharmacologic approaches, we investigated the role of EP2 receptor in two mouse models of ICH induced by intrastriatal injection of collagenase or autologous arterial whole blood. We used middle-aged male mice to enhance the clinical relevance of the study. EP2 receptor was expressed in neurons but not in astrocytes or microglia after collagenase-induced ICH. Brain injury after collagenase-induced ICH was associated with enhanced cellular and molecular inflammatory responses, oxidative stress, and matrix metalloproteinase (MMP)-2/9 activity. EP2 receptor deletion exacerbated brain injury, brain swelling/edema, neuronal death, and neurobehavioral deficits, whereas EP2 receptor activation by the highly selective agonist AE1-259-01 reversed these outcomes. EP2 receptor deletion also exacerbated brain edema and neurologic deficits in the blood ICH model. These findings support the premise that neuronal EP2 receptor activation by PGE2 protects brain against ICH injury in middle-aged mice through its anti-inflammatory and anti-oxidant effects and anti-MMP-2/9 activity. PGE2/EP2 signaling warrants further investigation for potential use in ICH treatment.