Expression of miR-200c corresponds with increased reactive oxygen species and hypoxia markers after transient focal ischemia in mice

Embolic stroke results in a necrotic core of cells destined to die, but also a peri-ischemic, watershed penumbral region of potentially salvageable brain tissue. Approaches to effectively differentiate between the ischemic and peri-ischemic zones is critical for novel therapeutic discovery to improve outcomes in survivors of stroke. MicroRNAs are a class of small non-coding RNAs regulating gene translation that have region- and cell-specific expression and responses to ischemia. We have previously reported that global inhibition of cerebral microRNA200c after experimental stroke in mice is protective, however delineating the post-stroke sub-regional and celltype specific patterns of post-stroke miR-200c expression are necessary to minimize off-target effects and advance translational application. Here, we detail a novel protocol to visualize regional miR-200c expression after experimental stroke, complexed with visualization of regional ischemia and markers of oxidative stress in an experimental stroke model in mice. In the present study we demonstrate that the fluorescent hypoxia indicator pimonidazole hydrochloride, the reactive-oxygen-species marker 8-hydroxy-deoxyguanosine, neuronal marker MAP2 and NeuN, and the reactive astrocyte marker GFAP can be effectively complexed to determine regional differences in ischemic injury as early as 30 min post-reperfusion after experimental stroke, and can be effectively used to distinguish ischemic core from surrounding penumbral and unaffected regions for targeted therapy. This multi-dimensional post-stroke immunofluorescent imaging protocol enables a greater degree of subregional mechanistic investigation, with the ultimate goal of developing more effective post-stroke pharmaceutical therapy.Peer reviewe

Ferroptosis Contributes to Isoflurane Neurotoxicity

The underlying mechanisms of isoflurane neurotoxicity in the developing brain remain unclear. Ferroptosis is a recently characterized form of programmed cell death distinct from apoptosis or autophagy, characterized by iron-dependent reactive oxygen species (ROS) generation secondary to failure of glutathione-dependent antioxidant defenses. The results of the present study are the first to demonstrate in vitro that ferroptosis is a central mechanism contributing to isoflurane neurotoxicity. We observed in embryonic mouse primary cortical neuronal cultures (day-in-vitro 7) that 6 h of 2% isoflurane exposure was associated with decreased transcription and protein expression of the lipid repair enzyme glutathione peroxidase 4. In parallel, isoflurane exposure resulted in increased ROS generation, disruption in mitochondrial membrane potential, and cell death. These effects were significantly attenuated by pre-treatment with the selective ferroptosis inhibitor ferrostatin-1 (Fer-1). Collectively, these observations provide a novel mechanism for isoflurane-induced injury in the developing brain and suggest that pre-treatment with Fer-1 may be a potential clinical intervention for neuroprotection

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Targeting Glial Mitochondrial Function for Protection from Cerebral Ischemia: Relevance, Mechanisms, and the Role of MicroRNAs

Astrocytes and microglia play crucial roles in the response to cerebral ischemia and are effective targets for stroke therapy in animal models. MicroRNAs (miRs) are important posttranscriptional regulators of gene expression that function by inhibiting the translation of select target genes. In astrocytes, miR expression patterns regulate mitochondrial function in response to oxidative stress via targeting of Bcl2 and heat shock protein 70 family members. Mitochondria play an active role in microglial activation, and miRs regulate the microglial neuroinflammatory response. As endogenous miR expression patterns can be altered with exogenous mimics and inhibitors, miR-targeted therapies represent a viable intervention to optimize glial mitochondrial function and improve clinical outcome following cerebral ischemia. In the present article, we review the role that astrocytes and microglia play in neuronal function and fate following ischemic stress, discuss the relevance of mitochondria in the glial response to injury, and present current evidence implicating miRs as critical regulators in the glial mitochondrial response to cerebral ischemia

Molecular Pathogenesis of Anti-NMDAR Encephalitis

Anti-NMDAR encephalitis is a recently identified autoimmune disease, described by an immune-mediated loss of NMDA glutamate receptors, resulting in progressive mental deterioration. To date, literature on anti-NMDAR encephalitis has been largely clinically oriented, including descriptions of the clinical presentation and course, diagnostic methods, and potential clinical treatments. However, the underlying molecular mechanisms contributing to the complex immunological cellular transformation that is associated with the progression of anti-NMDAR encephalitis remain to be adequately explored. This review will provide a summary of the current literature on anti-NMDAR encephalitis, including the immunologic molecular mechanisms contributing to disease progression. In particular this review will focus on the effect of anti-NMDAR on GluN2-NMDAR expression and the molecular transformation of B and T leukocytes in the loss of self-tolerance. Further research on the immunologic mechanisms contributing to anti-NMDAR encephalitis may provide an avenue for future novel diagnostic approaches, such as immunologic surveillance, as well as new therapeutic strategies for this recently identified autoimmune disease