T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice

T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion injury, but the relevant T cell–derived mediators of tissue injury remain unknown. Using a mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated in the injured mouse brain after cerebral ischemia. IL-21–deficient mice have smaller infarcts, improved neurological function, and reduced lymphocyte accumulation in the brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer experiments revealed that brain-infiltrating CD4+ T cells are the predominant IL-21 source. Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4+ T cells in the area surrounding acute stroke lesions, suggesting that IL-21–mediated brain injury may be relevant to human stroke

Stimulation of Na⁺/H⁺ Exchanger Isoform 1 Promotes Microglial Migration

Regulation of microglial migration is not well understood. In this study, we proposed that Na+/H+ exchanger isoform 1 (NHE-1) is important in microglial migration. NHE-1 protein was co-localized with cytoskeletal protein ezrin in lamellipodia of microglia and maintained its more alkaline intracellular pH (pHi). Chemoattractant bradykinin (BK) stimulated microglial migration by increasing lamellipodial area and protrusion rate, but reducing lamellipodial persistence time. Interestingly, blocking NHE-1 activity with its potent inhibitor HOE 642 not only acidified microglia, abolished the BK-triggered dynamic changes of lamellipodia, but also reduced microglial motility and microchemotaxis in response to BK. In addition, NHE-1 activation resulted in intracellular Na+ loading as well as intracellular Ca2+ elevation mediated by stimulating reverse mode operation of Na+/Ca2+ exchange (NCXrev). Taken together, our study shows that NHE-1 protein is abundantly expressed in microglial lamellipodia and maintains alkaline pHi in response to BK stimulation. In addition, NHE-1 and NCXrev play a concerted role in BK-induced microglial migration via Na+ and Ca2+ signaling. © 2013 Shi et al

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

The role of nicotinamide phosphoribosyltransferase in cerebral ischemia

As recombinant tissue plasminogen activator is the only drug approved for the clinical treatment of acute ischemic stroke, there is an urgent unmet need for novel stroke treatments. Endogenous defense mechanisms against stroke may hold the key to new therapies for stroke. A large number of studies suggest that nicotinamide phosphoribosyl-transferase (NAMPT) is an attractive candidate to improve post-stroke recovery. NAMPT is a multifunctional protein and plays important roles in immunity, metabolism, aging, inflammation, and stress responses. NAMPT exists in both the intracellular and extracellular space. As a rate-limiting enzyme, the intracellular form (iNAMPT) catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) from nicotinamide. iNAMPT closely regulates energy metabolism, enhancing the proliferation of endothelial cells, inhibiting apoptosis, regulating vascular tone, and stimulating autophagy in disease conditions such as stroke. Extracellular NAMPT (eNAMPT) is also known as visfatin (visceral fat–derived adipokine) and has pleotropic effects. It is widely believed that the diverse biological functions of eNAMPT are attributed to its NAMPT enzymatic activity. However, the effects of eNAMPT on ischemic injury are still controversial. Some authors have argued that eNAMPT exacerbates ischemic neuronal injury non-enzymatically by triggering the release of TNF-α from glial cells. In addition, NAMPT also participates in several pathophysiological processes such as hypertension, atherosclerosis, and ischemic heart disease. Thus, it remains unclear under what conditions NAMPT is beneficial or destructive. Recent work using in vitro and in vivo genetic/pharmacologic manipulations, including our own studies, has greatly improved our understanding of NAMPT. This review focuses on the multifaceted and complex roles of NAMPT under both normal and ischemic conditions

Demyelination as a rational therapeutic target for ischemic or traumatic brain injury

Previous research on stroke and traumatic brain injury (TBI) heavily emphasized pathological alterations in neuronal cells within gray matter. However, recent studies have highlighted the equal importance of white matter integrity in long-term recovery from these conditions. Demyelination is a major component of white matter injury and is characterized by loss of the myelin sheath and oligodendrocyte cell death. Demyelination contributes significantly to long-term sensorimotor and cognitive deficits because the adult brain only has limited capacity for oligodendrocyte regeneration and axonal remyelination. In the current review, we will provide an overview of the major causes of demyelination and oligodendrocyte cell death following acute brain injuries, and discuss the crosstalk between myelin, axons, microglia, and astrocytes during the process of demyelination. Recent discoveries of molecules that regulate the processes of remyelination may provide novel therapeutic targets to restore white matter integrity and improve long-term neurological recovery in stroke or TBI patients

Dietary supplementation with omega-3 polyunsaturated fatty acids robustly promotes neurovascular restorative dynamics and improves neurological functions after stroke

Stroke is a devastating neurological disease with no satisfactory therapies to preserve long-term neurological function, perhaps due to the sole emphasis on neuronal survival in most preclinical studies. Recent studies have revealed the importance of protecting multiple cell types in the injured brain, such as oligodendrocytes and components of the neurovascular unit, before long-lasting recovery of function can be achieved. For example, revascularization in the ischemic penumbra is critical to provide various neurotrophic factors that enhance the survival and activity of neurons and other progenitor cells, such as oligodendrocyte precursor cells. In the present study, we hypothesized that chronic dietary supplementation with fish oil promotes post-stroke angiogenesis, neurogenesis, and oligodendrogenesis, thereby leading to long-term functional improvements. Mice received dietary supplementation with n-3 PUFA-enriched fish oil for three months before and up to one month after stroke. As expected, dietary n-3 PUFAs significantly increased levels of n-3 PUFAs in the brain and improved long-term behavioral outcomes after cerebral ischemia. n-3 PUFAs also robustly improved revascularization and angiogenesis and boosted the survival of NeuN/BrdU labeled newborn neurons up to 35. days after stroke injury. Furthermore, these pro-neurogenic effects were accompanied by robust oligodendrogenesis. Thus, this is the first study to demonstrate that chronic dietary intake of n-3 PUFAs is an effective prophylactic measure not only to protect against ischemic injury for the long term but also to actively promote neurovascular restorative dynamics and brain repair

Oxidative stress and DNA damage after cerebral ischemia: Potential therapeutic targets to repair the genome and improve stroke recovery

The past two decades have witnessed remarkable advances in oxidative stress research, particularly in the context of ischemic brain injury. Oxidative stress in ischemic tissues compromises the integrity of the genome, resulting in DNA lesions, cell death in neurons, glial cells, and vascular cells, and impairments in neurological recovery after stroke. As DNA is particularly vulnerable to oxidative attack, cells have evolved the ability to induce multiple DNA repair mechanisms, including base excision repair (BER), nucleotide excision repair (NER) and non-homogenous endpoint jointing (NHEJ). Defective DNA repair is tightly correlated with worse neurological outcomes after stroke, whereas upregulation of DNA repair enzymes, such as APE1, OGG1, and XRCC1, improves long-term functional recovery following stroke. Indeed, DNA damage and repair are now known to play critical roles in fundamental aspects of stroke recovery, such as neurogenesis, white matter recovery, and neurovascular unit remodeling. Several DNA repair enzymes are essential for comprehensive neural repair mechanisms after stroke, including Pol? and NEIL3 for neurogenesis, APE1 for white matter repair, Gadd45b for axonal regeneration, and DNA-PKs for neurovascular remodeling. This review discusses the emerging role of DNA damage and repair in functional recovery after stroke and highlights the contribution of DNA repair to regenerative elements after stroke. This article is part of the Special Issue entitled ‘Cerebral Ischemia’

Ischemic preconditioning provides long-lasting neuroprotection against ischemic stroke: The role of Nrf2

Background and purpose: A major gap in the field of ischemic preconditioning (IPC) is whether or not long-lasting neuroprotection can be achieved. Moreover, the specific mechanisms underlying IPC and how they can be translated into the clinic remain uncertain. To fill these gaps, we tested the hypothesis that IPC exerts long-lasting structural and functional neuroprotection against ischemic stroke through the master gatekeeper of antioxidant defenses, nuclear factor erythroid 2-related factor 2 (Nrf2). We also tested whether the brain could be pharmaceutically preconditioned with a potent and blood-brain barrier-permeable Nrf2 activator, 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-trifluoethyl amide (CDDO-TFEA). Methods: IPC was induced by transient middle cerebral artery occlusion (MCAO) for 12 min, and ischemic stroke was generated by MCAO for 60 min in wild-type (WT) or Nrf2 knockout (KO) mice. Sensorimotor function, learning/memory skills, and brain tissue loss were measured up to 35 days after stroke. Primary rodent cortical neurons from wildtype (WT) and Nrf2 KO mice were subjected to lethal oxygen-glucose deprivation (OGD) or a brief OGD episode as a preconditioning (PC) stimulus before OGD. Cell viability/death, lipid electrophile generation, and Nrf2 activation were measured. CDDO-TFEA or its vehicle was administered in vivo for three consecutive days before MCAO. Tissue loss and neurological tests were performed 35 days after stroke. Results: IPC significantly reduced sensorimotor deficits, post-stroke cognitive impairments, and brain tissue loss, 35 days after MCAO in WT mice. These enduring protective effects of IPC were inhibited in Nrf2 KO mice. In neuronal cultures, PC also endowed primary neurons with ischemic tolerance against OGD-induced cell death, an effect that was abolished by loss of Nrf2 expression in KO neurons. PC induced the generation of low levels of lipid electrophiles and led to activation of the Nrf2 pathway. The mechanism underlying IPC may be translatable, as exogenous administration of the Nrf2 activator CDDO-TFEA significantly reduced neurological dysfunction and ischemic brain damage after MCAO. Conclusions: IPC provides long-lasting neuroprotection against ischemic brain injury and post-stroke cognitive dysfunction. Nrf2 activation plays a key role in this beneficial outcome and is a promising therapeutic target for the attenuation of ischemic brain injury

Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H(+) along with extracellular superoxide, and the intracellular H(+) must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na(+)/H(+) exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia-reperfusion was likewise prevented by inhibiting Na(+)/H(+) exchange and by reduced expression of the Na(+)/H(+) exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na(+)/H(+) exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production