Sarah L. Milton

Brain Na^+/K^+-ATPase activity in two anoxia tolerant vertebrates : Crucian carp and freshwater turtle

The crucian carp (Carassius carassius) and freshwater turtles (Trachemys scripta) are among the very few vertebrates that can survive extended periods of anoxia. The major problem for an anoxic brain is energy deficiency. In the brain, the Na+/K+-ATPase is the single most ATP consuming enzyme, being responsible for maintaining ion gradients. We here show that the Na+/K+-ATPase activity in the turtle brain is reduced by 31% in telencephalon and by 34% in cerebellum after 24 h of anoxia. Both changes were reversed upon reoxygenation. By contrast, the Na+/K+-ATPase activities were maintained in the anoxic crucian carp brain. These results support the notion that crucian carp and turtles use divergent strategies for anoxic survival. The fall in Na+/K+-ATPase activities displayed by the turtle is likely to be related to the strong depression of brain electric and metabolic activity utilized as an anoxic survival strategy by this species.

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. By contrast, Hsc73 was progressively increased throughout 12 hours of anoxia. This differential expression suggests different protective roles: Hsp72 in the initial downregulatory transition phase, and Hsc73 in maintaining neural network integrity during the long-term hypometabolic phase.

No oxygen? No problem! Intrinsic brain tolerance to hypoxia in vertebrates.

Many vertebrates are challenged by either chronic or acute episodes of low oxygen availability in their natural environments. Brain function is especially vulnerable to the effects of hypoxia and can be irreversibly impaired by even brief periods of low oxygen supply. This review describes recent research on physiological mechanisms that have evolved in certain vertebrate species to cope with brain hypoxia. Four model systems are considered: freshwater turtles that can survive for months trapped in frozen-over lakes, arctic ground squirrels that respire at extremely low rates during winter hibernation, seals and whales that undertake breath-hold dives lasting minutes to hours, and naked mole-rats that live in crowded burrows completely underground for their entire lives. These species exhibit remarkable specializations of brain physiology that adapt them for acute or chronic episodes of hypoxia. These specializations may be reactive in nature, involving modifications to the catastrophic sequelae of oxygen deprivation that occur in non-tolerant species, or preparatory in nature, preventing the activation of those sequelae altogether. Better understanding of the mechanisms used by these hypoxia-tolerant vertebrates will increase appreciation of how nervous systems are adapted for life in specific ecological niches as well as inform advances in therapy for neurological conditions such as stroke and epilepsy.

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. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3‐dihydroxybenzoic acid (2,3‐DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H2O2 increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro, while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H2O2), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.

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. In the anoxic turtle brain the inhibitory tone is strengthened through a sustained rise in extracellular GABA, and a corresponding increase in the density of GABA(A) receptors. The turtle has enhanced mechanisms that protect against the formation of ROS and mechanisms to protect from ROS damage. As many of these may be selectively activated during anoxia and recovery, the turtle could serve as a useful model to identify and investigate mechanisms for activating key protection and rescue mechanisms implicated in aging.

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. Levels of the ER stress protein Grp78 were unchanged. Stress proteins remained elevated in long‐term anoxia while the Bcl‐2/Bax ratio was unaltered. No changes in cleaved caspase 3 levels were observed during anoxia while apoptosis inducing factor increased significantly. Furthermore, we found no evidence for the anoxic translocation of Bax from the cytosol to mitochondria, nor movement of apoptosis inducing factor between the mitochondria and nucleus. Reoxygenation did not lead to further increases in stress proteins or apoptotic regulators except for HO‐1. The apparent protection against cell damage was corroborated with immunohistochemistry, which indicated no overt damage in the turtle brain subjected to anoxia and reoxygenation. The results suggest that molecular adaptations enhance pro‐survival mechanisms and suppress apoptotic pathways to confer anoxia tolerance in freshwater turtles.

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. Phosphorylated ERK1/2 (p-ERK) and AKT (p-AKT) as well as Bcl-2 are upregulated in anoxic neuronally enriched primary cultures from turtle brain. This native upregulation is further increased by the selective A1R agonist 2-chloro-N-cyclopentyladenosine (CCPA), whereas the selective antagonist 8-cyclopentyl-1,3-dihydropylxanthine (DPCPX) decreases p-ERK and p-AKT expression. Conversely, A1R antagonism resulted in increases in phosphorylated JNK (p-JNK), p38 (p-p38), and Bax. As pathological and adaptive changes occur simultaneously during anoxia/ischemia in mammalian neurons, the turtle provides an alternative model to analyze protective mechanisms in the absence of evident pathologies.

Post-transcriptional gene silencing by RNA interference in non-mammalian vertebrate systems: Where do we stand?

RNA interference (RNAi), the process by which double stranded RNA induces the silencing of endogenous genes through the degradation of its correspondent messenger RNA, has been used for post-transcriptional gene silencing allowing scientists to better understand gene function, becoming a powerful tool in reverse genetics for in vivo and in vitro systems. Successful results in vivo have been obtained from invertebrate animal models, whereas vertebrate systems have been limited primarily to mammalian models and cell lines. Nevertheless, exciting results have also been reported from non-mammalian vertebrate models, such as the knock-down of endogenous genes in Xenopus tadpoles by a construct containing both a Xenopus-specific shRNA sequence and the human Ago2 (which is a key enzyme in the RNAi silencing complex), or the design of a novel vector expressing a miRNA driven by a tissue-specific promoter in zebrafish, and the use of an avian retroviral vector to deliver miRNA and shRNA in chicken embryos proving to be effective in knocking-down endogenous genes with a long lasting effect, to mention some examples. Whether dsRNA is able to initiate a specific RNAi response, or all the factors required for RNAi are present in non-mammalian vertebrates, are still questions which remain to be answered. Further progress in understanding natural RNAi mechanisms in non-mammalian vertebrates will help scientists to overcome difficulties and improve this gene silencing technology. There is no doubt that in few years RNAi silencing approaches will become the tool of choice to knock-down genes in all groups of non-mammalian vertebrates, fulfilling different purposes, from basic research to animal therapeutics and drug discovery.

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. Ngb expression is strongly up‐regulated by hypoxia and post‐anoxia reoxygenation but increases only modestly in anoxia. The potential neuroprotective role of Ngb in this species was analyzed by knocking down Ngb using specific small interfering RNA. Ngb knockdown in neuronally enriched cell cultures resulted in significant increases in H2O2 release compared to controls but no change in cell death. Cell survival may be linked to activation of other protective responses such as the extracellular regulated kinase transduction pathway, as phosphorylated extracellular regulated kinase levels in anoxia were significantly higher in Ngb knockdown cultures compared to controls. The greater expression of Ngb when reactive oxygen species are likely to be high, and the increased susceptibility of neurons to H2O2 release and external oxidative stress in knockdown cultures, suggests a role for Ngb in reducing reactive oxygen species production or in detoxification, though it does not appear to be of primary importance in the anoxia tolerant turtle in the presence of compensatory survival mechanisms.

Low extracellular dopamine levels are maintained in the anoxic turtle (Trachemys scripta) striatum.

The uncontrolled increase of extracellular dopamine (DA) has been implicated in the pathogenesis of hypoxic/ischemic damage in the mammalian brain. But unlike the harmful release of excitatory neurotransmitters such as glutamate and aspartate, which occurs on brain depolarization, excessive extracellular DA levels occur even with mild hypoxia in the mammalian brain. The purpose of this study was to determine whether hypoxia/anoxia provokes a similar increase in the anoxic tolerant turtle brain. Extracellular DA was measured in the striatum of the turtle using microdialysis followed by high-performance liquid chromatography analysis. Results show that extracellular DA was held to normoxic levels over 4 hours of anoxia. Treatment with the specific DA transport blocker GBR 12909 during anoxia resulted in a significant increase in DA to 236% over basal levels. The ability to maintain low striatal extracellular DA may be an important adaptation for anoxic survival in the turtle brain; a contributing factor is the continued functioning of DA uptake mechanisms during anoxia.