[show abstract][hide abstract] ABSTRACT: 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.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 02/2011; 31(2):467-75. · 5.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Neurochemistry 07/2009; 109(5):1413-26. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 H(2)O(2) 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 H(2)O(2) 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.
Journal of Neurochemistry 06/2009; 110(2):603-12. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Postnatal neurogenesis in response to stroke or ischemia is currently of great medical interest. In this study, we investigated the potential for neurogenesis in an anoxia tolerant vertebrate in response to global ischemia. The results suggest sustained neurogenesis in the turtle that increases after ischemic damage, thus revealing a potential physiological adaptation to repeated anoxia–reoxygenation events. This finding further emphasizes the common vertebrate phenomenon of postnatal neurogenesis, with the capacity for extensive regeneration of neurons apparent in some reptilian species.
[show abstract][hide abstract] ABSTRACT: To survive anoxia, neural ATP levels have to be defended. Reducing electrical activity, which accounts for 50% or more of neural energy consumption, should be beneficial for anoxic survival. The retina is a hypoxia sensitive part of the central nervous system. Here, we quantify the in vivo retinal light response (electroretinogram; ERG) in three vertebrates that exhibit varying degrees of anoxia tolerance: freshwater turtle (Trachemys scripta), epaulette shark (Hemiscyllium ocellatum) and leopard frog (Rana pipiens). A virtually total suppression of ERG in anoxia, probably resulting in functional blindness, has previously been seen in the extremely anoxia-tolerant crucian carp (Carassius carassius). Surprisingly, the equally anoxia-tolerant turtle, which strongly depresses brain and whole-body metabolism during anoxia, exhibited a relatively modest anoxic reduction in ERG: the combined amplitude of turtle ERG waves was reduced by approximately 50% after 2 h. In contrast, the shark b-wave amplitude practically disappeared after 30 min of severe hypoxia, and the frog b-wave was decreased by approximately 75% after 40 min in anoxia. The specific A(1) adenosine receptor antagonist CPT significantly delayed the suppression of turtle ERG, while the hypoxic shark ERG was unaffected by the non-specific adenosine receptor antagonist aminophylline, suggesting adenosinergic involvement in turtle but not in shark.
Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 09/2008; 150(4):395-403. · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. ERK1/2 and Akt were significantly upregulated during the first hour of transition to full anoxia, but returned to baseline by 4 h anoxia. Conversely, p38MAPK levels were suppressed by a mean 71% at 1 h anoxia but also returned to baseline by 4 h anoxia. Systemic administration of the general adenosine receptor antagonist aminophylline abrogated the increases in both phosphorylated ERK1/2 and Akt, as well as the initial suppression of p38MAPK. The differential modulation of the MAPK/Akt pathways may be critical for neuronal protection during the initial transition to the hypometabolic state during anoxia, when physiologic stress is likely to be greatest.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 05/2008; 28(8):1469-77. · 5.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. Our attention has recently turned to molecular mechanisms of anoxia tolerance, including the upregulation of such protective factors as heat shock proteins (Hsp72, Hsc73), the reversible downregulation of voltage gated potassium channels, and the modulation of MAP kinase pathways. In this review we discuss three phases of anoxia tolerance, including the initial metabolic downregulation over the first several hours, the long-term maintenance of neuronal function over days to weeks of anoxia, and finally recovery upon reoxygenation, with necessary defenses against reactive oxygen stress.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 07/2007; 147(2):277-90. · 2.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 H(2)O(2) 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 (H(2)O(2)), 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.
Journal of Neurochemistry 06/2007; 101(4):993-1001. · 3.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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. However, the regulation of Neuroglobin (Ngb) under conditions of varying oxygen is controversial. This study examines the regulation of Ngb in an anoxia-tolerant vertebrate under conditions of hypoxia and anoxia. The freshwater turtle Trachemys scripta can withstand complete anoxia for days, and adaptations that permit neuronal survival have been extensively examined. Turtle neuroglobin specific primers were employed in RT-PCR for determining the regulation of neuroglobin mRNA expression in turtles placed in normoxia, hypoxia (4 h), anoxia (1 and 4 h), and anoxia-reoxygenation. Whole brain expression of neuroglobin is strongly upregulated by hypoxia and post-anoxic-reoxygenation in T. scripta, with a lesser degree of upregulation at 1 and 4 h anoxia. Our data implicate neuroglobin in mediating brain anoxic survival.
Journal of Biomedical Science 08/2006; 13(4):509-14. · 2.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Excessive dopamine (DA) is known to cause hypoxic/ischemic damage to mammalian brain. The freshwater turtle Trachemys scripta, however, maintains basal striatal DA levels in anoxia. We investigated DA balance during early anoxia when energy status in the turtle brain is compromised. The roles of ATP-sensitive potassium (K(ATP)) channels and adenosine (AD) receptors were investigated as these factors affect DA balance in mammalian neurons. Striatal extracellular DA was determined by microdialysis with HPLC in the presence or absence of the specific DA transport blocker GBR-12909, the K(ATP) blocker 2,3-butanedione monoxime, or the nonspecific AD receptor blocker theophylline. We found that in contrast to long-term anoxia, blocking DA reuptake did not significantly increase extracellular levels in 1-h anoxic turtles. Low DA levels in early anoxia were maintained instead by activation of K(ATP) channels and AD receptors. Blocking K(ATP) resulted in a 227% increase in extracellular DA in 1-h anoxic turtles but had no effect after 4 h of anoxia. Similarly, blocking AD receptors increased DA during the first hour of anoxia but did not change DA levels at 4-h anoxia. Support for the role of K(ATP) channels in DA balance comes from normoxic animals treated with K(ATP) opener; infusing diazoxide but not adenosine into the normoxic turtle striatum resulted in an immediate DA decrease to 14% of basal values within 1.5 h. Alternative strategies to maintain low extracellular levels may prevent catastrophic DA increases when intracellular energy is compromised while permitting the turtle to maintain a functional neuronal network during long-term anoxia.
[show abstract][hide abstract] ABSTRACT: The turtle brain's extraordinary ability to tolerate anoxia is based on constitutive and expressed factors. Constitutive factors that predispose for anoxia tolerance include enhanced levels of glycogen stores, increased densities of protective receptors, elevated antioxidant capacities and elevated heat shock protein. However, to survive an anoxic insult, three distinct phases must be negotiated successfully. (1) A coordinated downregulation of ATP demand processes to basal levels. This phase, which takes 1-2 h, includes a reduction in voltage-gated K(+) (Kv) channel transcription and a substantial increase in Hsp72 and Hsc73 levels. During this period, adenosine and K(ATP) channels mediate several key events including channel arrest initiation and a reduction in the release of excitatory amino acids (EAAs). (2) Long-term survival (days) at basal levels of ATP expenditure. Neuronal network integrity is preserved through the continued operation of core activities. These include periodic electrical activity, an increased release of GABA and a continued release of glutamate and dopamine. Adenosine and GABA modulate the glutamate release. There is a further increase in Hsc73, indicating a 'housekeeping' role for this protein during this period. (3) A rapid upregulation of neuronal processes when oxygen becomes available to restore full function, together with the activation of protection mechanisms against reperfusion-generated reactive oxygen species.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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. We found that in turtle brains exposed to 4-h anoxia Kv1 transcripts were reduced to 18.5% of normoxic levels. Kv1 channel mRNA levels were restored to normal within 4 h of subsequent reoxygenation. Our results provide clear evidence that brain Kv channel expression is sensitive to oxygen supply and indicate an important mechanism that matches brain activity to oxygen supply.
[show abstract][hide abstract] ABSTRACT: While frogs such as Rana temporaria are known to withstand 4-5 h anoxia at room temperature, little is known about the neurological adaptations that permit this. Previous research has shown that changes in neuroactive compounds such as glutamate and dopamine in anoxia-sensitive (mammalian) brains follow a strikingly different pattern than is observed in truly anoxia-tolerant vertebrates such as the freshwater turtle. The present study measured changes in the levels of whole brain and extracellular amino acids, and extracellular dopamine, in the normoxic and 3-4 h anoxic frog Rana pipiens, in order to determine whether their neurotransmitter responses resemble the anoxia-vulnerable or anoxia-tolerant response. Increases in whole brain serine, glycine, alanine and GABA levels were similar to those seen in anoxia-tolerant species, although the levels of glutamine, taurine and glutamate did not increase as occurs in true facultative anaerobes. Extracellular levels of aspartate, taurine and GABA also increased significantly, while glutamate levels decreased. The maintenance of low extracellular glutamate was the most significant difference between the frog and the anoxic/ischemic mammalian brain, although aspartate did increase 215% over a 4 h period of anoxia. A 12-fold increase in extracellular dopamine levels during anoxia was the biggest contrast between anoxia-tolerant vertebrates and R. pipiens. The frog could thus be an interesting model in which to examine the mechanisms of dopamine failure in early anoxia, which occurs rapidly in the mammal but over a period of hours in the 'slow death' of the anoxic frog brain.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The turtle Trachemys scripta is one of a limited group of vertebrates that can withstand hours to days without oxygen. One facet of anoxic survival is the turtle's ability to maintain basal extracellular glutamate levels, whereas in most vertebrates, anoxia triggers massive excitotoxic glutamate release. We investigated glutamate release and reuptake in the anoxic turtle and the effects of adenosine and ATP-sensitive potassium (K(ATP)) channels on glutamate homeostasis. Striatal extracellular glutamate was measured in anesthetized T. scripta by microdialysis in normoxia and over 2-h anoxia. Glutamate release is decreased by 44% in the early anoxic turtle; this anoxia-induced decrease in glutamate release was prevented when K(ATP) channels and adenosine receptors were blocked simultaneously but not when either mechanism was blocked individually. We hypothesize that the continued release and reuptake of glutamate during anoxia help maintain neuronal tone and aid in the recovery of a functional neuronal network after long periods of anoxia, whereas activation of adenosine and/or K(ATP) conserves energy by reducing glutamate release and lowering transport costs.
[show abstract][hide abstract] ABSTRACT: Because of the close overlap of hurricane season (June-November) and sea turtle nesting season (March-November) in the Caribbean and Northwest Atlantic Oceans, hurricanes are a potential cause of damage to sea turtle populations. However, no data has been gathered on either the immediate or long term effects of hurricanes on adult sea turtles or their nesting beaches. Hurricane Andrew, which struck South Florida on 24 August 1992, provided a unique opportunity to quantify the impact of a category 4 hurricane on six Florida nesting beaches. It was determined that Hurricane Andrew affected turtle nests over a total of 90 miles of beaches on the east and west coasts of Florida. We found that the storm surge associated with the hurricane produced the greatest mortality through nest flooding. The greatest surge effect was felt on beaches closest to the “eye” of the hurricane, where egg mortality was 100%. In areas farther away from the “eye,” the surge was lower and mortality was correspondingly decreased. Detailed data on post-hurricane hatching success, mortality, and cause of death was gathered on eight relocated and eight in situ nests on Fisher Island in Miami, Florida, which suffered from flooding and extensive changes in topography. Sixty-nine percent of the eggs did not hatch after Hurricane Andrew and appeared to have drowned during the storm. Further mortality occurred when surviving turtles suffocated in nests situated in the beach zone where sand had accreted. This later mortality may be substantially reduced if beach topography is returned to normal and beach debris removed after a hurricane.