The Role of NMDA Receptor Partial Antagonist, Carbamathione, as a Therapeutic Agent for Transient Global Ischemia.

Carbamathione (Carb), an NMDA glutamate receptor partial antagonist, has potent neuroprotective functions against hypoxia- or ischemia-induced neuronal injury in cell- or animal-based stroke models. We used PC-12 cell cultures as a cell-based model and bilateral carotid artery occlusion (BCAO) for stroke. Whole-cell patch clamp recording in the mouse retinal ganglion cells was performed.

A novel sulindac derivative protects against oxidative damage by a cyclooxygenase-independent mechanism.

Oxidative damage is believed to play a major role in the etiology of many age-related diseases and the normal aging process. We previously reported that sulindac, a cyclooxygenase (COX) inhibitor and FDA approved anti-inflammatory drug, has chemoprotective activity in cells and intact organs by initiating a pharmacological preconditioning response, similar to ischemic preconditioning (IPC). The mechanism is independent of its COX inhibitory activity as suggested by studies on the protection of the heart against oxidative damage from ischemia/reperfusion and retinal pigmented endothelial (RPE) cells against chemical oxidative and UV damage Unfortunately, sulindac is not recommended for long-term use due to toxicities resulting from its COX inhibitory activity.

The expression of genes involved in excitatory and inhibitory neurotransmission in turtle (Trachemys scripta) brain during anoxic submergence at 21 °C and 5 °C reveals the importance of cold as a preparatory cue for anoxia survival.

We investigated if transcriptional responses are consistent with the arrest of synaptic activity in the anoxic turtle (Trachemys scripta) brain. Thirty-nine genes of key receptors, transporters, enzymes and regulatory proteins of inhibitory and excitatory neurotransmission were partially cloned and their expression in telencephalon of 21 °C- and 5 °C-acclimated normoxic, anoxic (24 h at 21 °C; 1 and 14 days at 5 °C) and reoxygenated (24 h at 21 °C; 13 days at 5 °C) turtles quantified by real-time RT-PCR. Gene expression was largely sustained with anoxia at 21 °C and 5 °C.

Cell-specific gene therapy driven by an optimized hypoxia-regulated vector reduces choroidal neovascularization.

Unlabelled: Aberrant growth of blood vessels in the choroid layer of the eye, termed choroidal neovascularization (CNV), is the pathological hallmark of exudative age-related macular degeneration (AMD), causing irreversible blindness among the elderly. Co-localization of proangiogenic factors and hypoxia inducible factors (HIF) in neovascular membranes from AMD eyes suggests the role of hypoxia in pathogenesis of CNV. In order to utilize hypoxic conditions in RPE for therapeutic purposes, we developed an optimized hypoxia regulated, RPE cell-specific gene therapy to inhibit choroidal neovascularization.

Lessons from nature: signalling cascades associated with vertebrate brain anoxic survival.

What is the topic of this review? Although the mammalian brain is exquisitely sensitive to hypoxia, some turtles survive complete anoxia by decreasing metabolic demand to match reduced energy supply. These animal models may help to elucidate neuroprotective mechanisms and reveal novel therapeutic targets for diseases of oxygen deprivation. What advances does it highlight? The mitogen-activated protein kinases (MAPKs) are part of the suite of adaptive responses to anoxia that are modulated by adenosine, a 'retaliatory metabolite' released in early anoxia.

A hypoxia-responsive glial cell-specific gene therapy vector for targeting retinal neovascularization.

Purpose: Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model.

Upregulation of Hsp72 mediates anoxia/reoxygenation neuroprotection in the freshwater turtle via modulation of ROS.

The neuroprotective role of Hsp72 has been demonstrated in several ischemic/stroke models to occur primarily through mediation of apoptotic pathways, and a number of heat shock proteins are upregulated in animal models capable of extended anoxic survival. In the present study, we investigated the role of Hsp72 on cell death and apoptotic regulators in one anoxia tolerant model system, the freshwater turtle Trachemys scripta. Since Hsp72 is known to regulate apoptosis through interactions with Bcl-2, we manipulated the levels of Hsp72 and Bcl-2 with siRNA in neuronally enriched primary cell cultures and examined downstream effects.

Characterization of electroencephalographic and biochemical responses at 5-HT promoting drug-induced onset of serotonin syndrome in rats.

Many psychotropic substances used either for medications or illicit recreational purposes are able to produce an increase in extracellular serotonin (5HT) in the CNS. 5HT is well known to improve mood; however, only when the levels of its release are in an appropriate range. Excessive 5HT is harmful, and will generally result in serotonin syndrome.

Hypoxia-regulated retinal glial cell-specific promoter for potential gene therapy in disease.

Purpose: Retinal Müller cells span the retina and secrete several trophic factors and represent the functional link between blood vessels and neurons, making them attractive targets for gene therapy. Therefore, a hypoxia-regulated, retinal glial cell-specific vector was constructed and tested for its response to hypoxia.

Methods: A hybrid promoter containing domains of human glial fibrillary acidic protein (GFAP) and several hypoxia-responsive and aerobically silenced elements (HRSE) was incorporated separately into plasmid vectors for generation of self-complementary adeno-associated virus.

Neuroprotective signaling pathways are modulated by adenosine in the anoxia tolerant turtle.

Cumulative evidence shows a protective role for adenosine A1 receptors (A1R) in hypoxia/ischemia; A1R stimulation reduces neuronal damage, whereas blockade exacerbates damage. The signal transduction pathways may involve the mitogen-activated protein kinase (MAPK) pathways and serine/threonine kinase (AKT), with cell survival depending on the timing and degree of upregulation of these cascades as well as the balance between pro-survival and pro-death pathways. Here, we show in vitro that extracellular signal-regulated kinase (ERK1/2) and phosphatidylinositol 3-kinase (PI3-K/AKT) activation is dependent on A1R stimulation, with further downstream effects that promote neuronal survival.

Modulation of stress proteins and apoptotic regulators in the anoxia tolerant turtle brain.

Freshwater turtles survive prolonged anoxia and reoxygenation without overt brain damage by well-described physiological processes, but little work has been done to investigate the molecular changes associated with anoxic survival. We examined stress proteins and apoptotic regulators in the turtle during early (1 h) and long-term anoxia (4, 24 h) and reoxygenation. Western blot analyses showed changes within the first hour of anoxia; multiple stress proteins (Hsp72, Grp94, Hsp60, Hsp27, and HO-1) increased while apoptotic regulators (Bcl-2 and Bax) decreased.

Role of neuroglobin in regulating reactive oxygen species in the brain of the anoxia-tolerant turtle Trachemys scripta.

Neuroglobin (Ngb) is an oxygen binding heme protein found in nervous tissue with a yet unclear physiological and protective role in the hypoxia-sensitive mammalian brain. Here we utilized in vivo and in vitro studies to examine the role of Ngb in anoxic and post-anoxic neuronal survival in the freshwater turtle. We employed semiquantitative RT-PCR and western blotting to analyze Ngb mRNA and protein levels in turtle brain and neuronally enriched cultures.

Adenosine modulates ERK1/2, PI3K/Akt, and p38MAPK activation in the brain of the anoxia-tolerant turtle Trachemys scripta.

The fate of cells under anoxic or ischemic stress is determined by intracellular signaling pathways including the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI3K/Akt), which affect downstream members of the apoptotic cascade. The freshwater turtle Trachemys scripta is extremely tolerant of anoxia, surviving up to 48 h at room temperature and for weeks at 3 degrees C in the complete absence of oxygen. We investigated the relationship between the neuroprotective purine adenosine, which increases greatly in the anoxic turtle brain, and MAPK and Akt activation during both short (1 h) and long-term (4 h) anoxia.

Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector.

Purpose: To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye.

Methods: Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65).

Regulation of extracellular glutamate levels in the long-term anoxic turtle striatum: coordinated activity of glutamate transporters, adenosine, K (ATP) (+) channels and GABA.

Early in anoxia the mammalian brain experiences an uncontrolled release of glutamate, which combined with the failure of glutamate reuptake mechanisms, leads to massive neurotoxic increases in extracellular glutamate. By contrast, the anoxia tolerant turtle (Trachemys scripta) shows no increase in extracellular glutamate levels over many hours of anoxia. During the first hours of anoxia extracellular glutamate levels are maintained by a reduction in glutamate release (mainly due to the inhibition of neuronal vesicular glutamate release), combined with continued uptake by still active glutamate transporters.

Role of myofibril-inducing RNA in cardiac TnT expression in developing Mexican axolotl.

The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene "c", for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts. Myofibril-inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter.

Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta.

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS.

Beyond anoxia: the physiology of metabolic downregulation and recovery in the anoxia-tolerant turtle.

The freshwater turtle Trachemys scripta is among the most anoxia-tolerant of vertebrates, a true facultative anaerobe able to survive without oxygen for days at room temperature to weeks or months during winter hibernation. Our good friend and colleague Peter Lutz devoted nearly 25 years to the study of the physiology of anoxia tolerance in these and other model organisms, promoting not just the basic science but also the idea that understanding the physiology and molecular mechanisms behind anoxia tolerance provides insights into critical survival pathways that may be applicable to the hypoxic/ischemic mammalian brain. Work by Peter and his colleagues focused on the factors which enable the turtle to enter a deep hypometabolic state, including decreases in ion flux ("channel arrest"), increases in inhibitory neuromodulators like adenosine and GABA, and the maintenance of low extracellular levels of excitatory compounds such as dopamine and glutamate.

Gene transcription of neuroglobin is upregulated by hypoxia and anoxia in the brain of the anoxia-tolerant turtle Trachemys scripta.

Neuroglobin is a heme protein expressed in the vertebrate brain in mammals, fishes, and birds. The physiological role of neuroglobin is not completely understood but possibilities include serving as an intracellular oxygen-carrier or oxygen-sensor, as a terminal oxidase to regenerate NAD(+) under anaerobic conditions, or involvement in NO or ROS metabolism. As the vertebrate nervous system is particularly sensitive to hypoxia, an intracellular protein that helps sustain cellular respiration would aid hypoxic survival.

The upregulation of cognate and inducible heat shock proteins in the anoxic turtle brain.

Because heat shock proteins (HSPs) have an important protective function against ischemia/anoxia in mammalian brain, the authors investigated the expression of Hsp72 and Hsc73 in the anoxia-surviving turtle brain. Unlike the mammalian brain, high levels of Hsp72 were found in the normoxic turtle brain. Hsp72 levels were significantly increased by 4 hours of anoxia, remained constant until 8 hours, and then decreased to baseline at 12 hours.

Gene transcription of brain voltage-gated potassium channels is reversibly regulated by oxygen supply.

Voltage-dependent potassium channels (Kv channels) are important determinants of brain electrical activity. Hypoxia may be an important modifier, because several voltage-gated K+ channels are reversibly blocked by acute hypoxia and are thought to act as oxygen sensors. Here we show, using the anoxia-tolerant turtle brain (Trachemys scripta) as a model, that brain Kv1 channel transcription is reversibly regulated by oxygen supply.

Is turtle longevity linked to enhanced mechanisms for surviving brain anoxia and reoxygenation?

We suggest that the processes that protect the turtle brain against anoxia and subsequent reoxygenation might also contribute to turtle longevity since many of them are linked to age related neurodegeneration. In the turtle the mechanisms for conserving ion channel function are particularly robust. The anoxic turtle brain avoids excitatory neurotransmitter toxicity by maintaining a balance between dopamine and glutamate-release and still active uptake mechanisms.