Faridis Serrano

Reactive oxygen species and synaptic plasticity in the aging hippocampus.

Aging is associated with a general decline in physiological functions including cognitive functions. Given that the hippocampus is known to be critical for certain forms of learning and memory, it is not surprising that a number of neuronal processes in this brain area appear to be particularly vulnerable to the aging process. Long-term potentiation (LTP), a form of synaptic plasticity that has been proposed as a biological substrate for learning and memory, has been used to examine age-related changes in hippocampal synaptic plasticity. A current hypothesis states that oxidative stress contributes to age-related impairment in learning and memory. This is supported by a correlation between age, memory impairment, and the accumulation of oxidative damage to cellular macromolecules. However, it also has been demonstrated that ROS are necessary components of signal transduction cascades during normal physiological processes. This review discusses the evidence supporting the dual role of reactive oxygen species (ROS) as cellular messenger molecules in normal LTP, as well their role as damaging toxic molecules in the age-related impairment of LTP. In addition, we will discuss parallel analyses of LTP and behavioral tests in mice that overexpress antioxidant enzymes and how the role of antioxidant enzymes and ROS in modulating these processes may vary over the lifespan of an animal.

NADPH oxidase immunoreactivity in the mouse brain

Superoxide production via NADPH oxidase has been shown to play a role in neurotoxicity, ischemic stroke, and possibly Parkinsons and Alzheimers diseases. In addition, NADPH oxidase-dependent production of superoxide may be necessary for normal brain functions, including neuronal differentiation and neuronal plasticity. To improve our understanding of NADPH oxidase in the brain, we studied the localization of the various protein components of NADPH oxidase in the central nervous system of the adult mouse using immunohistochemistry. We detected staining for the cytoplasmic NADPH proteins, p40(phox), p47(phox), and p67(phox), as well as the membrane-associated NADPH oxidase proteins, p22(phox) and gp91(phox) in neurons throughout the mouse brain. Staining of each of the NADPH oxidase proteins was observed in neurons in all regions of the neuraxis, with particularly prominent localizations in the hippocampus, cortex, amygdala, striatum, and thalamus. The expression of NADPH oxidase proteins in neurons suggests the possibility that enzymatic production of superoxide by a NADPH oxidase may play a role in both normal neuronal function as well as neurodegeneration in the brain.

Synaptic localization of a functional NADPH oxidase in the mouse hippocampus.

Superoxide has been shown to be critical for hippocampal long-term potentiation (LTP) and hippocampus-dependent memory function. A possible source for the generation of superoxide during these processes is NADPH oxidase. The active oxidase consists of two membrane proteins, gp91phox and p22phox, and four cytosolic proteins, p40phox, p47phox, p67phox, and Rac. Upon stimulation, the cytosolic proteins translocate to the membrane to form a complex with the membrane components, which results in production of superoxide. Here, we determined the presence, localization, and functionality of a NADPH oxidase in mouse hippocampus by examining the NADPH oxidase proteins as well as the production of superoxide. All of the NADPH oxidase proteins were present in hippocampal homogenates and enriched in synaptoneurosome preparations. Immunocytochemical analysis of cultured hippocampal neurons indicated that all NADPH oxidase proteins were localized in neuronal cell bodies as well as dendrites. Furthermore, double labeling analysis using antibodies to p67phox and the presynaptic marker synaptophysin suggest a close association of the NADPH oxidase subunits with synaptic sites. Finally, stimulation of hippocampal slices with phorbol esters triggered translocation of the cytoplasmic NADPH oxidase proteins to the membrane and an increase in superoxide production that was blocked by inhibitors of NADPH oxidase. Taken together, our data suggest that NADPH oxidase is present in mouse hippocampus and might be the source of superoxide production required for LTP and memory function.

Aging-Dependent Alterations in Synaptic Plasticity and Memory in Mice That Overexpress Extracellular Superoxide Dismutase

Oxidative damage caused by reactive oxygen species (ROS) has been proposed to be critically involved in several pathological manifestations of aging, including cognitive dysfunction. ROS, including superoxide, are generally considered as neurotoxic molecules whose effects can be alleviated by antioxidant enzymes. However, ROS also are known to be necessary components of the signal transduction cascades underlying normal synaptic plasticity. Therefore, we reasoned that the role that ROS and antioxidant enzymes play in modulating neuronal processes varies over the lifespan of an animal. We examined hippocampal long-term potentiation (LTP) and memory-related behavioral performance in transgenic mice overexpressing extracellular superoxide dismutase (EC-SOD) and their wild-type littermates at different ages. We found that aged EC-SOD transgenic mice exhibited enhanced hippocampal LTP, better cerebellum-dependent motor learning, and better hippocampus-dependent spatial learning compared with their wild-type littermates. We also found that EC-SOD overexpression impaired contextual learning, but the impairment was decreased in the aged transgenic mice. At the molecular level, aged EC-SOD transgenic mice had lower superoxide levels, a decrease in protein carbonyl levels, and a decrease in p38 and extracellular signal-regulated kinase 2 phosphorylation compared with aged wild-type mice. Our findings suggest that elevated levels of superoxide contribute to aging-related impairments in hippocampal LTP and memory, and that these impairments can be alleviated by overexpression of EC-SOD. We conclude that there is an age-dependent alteration in the role of superoxide in modulating synaptic plasticity and learning and memory.

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice.

Background While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits. Research Design and Methods T1-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively. Results STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged. Conclusion Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.

Brain preservation with selective cerebral perfusion for operations requiring circulatory arrest: protection at 25 °C is similar to 18 °C with shorter operating times

BACKGROUND Hypothermic circulatory arrest (HCA) is employed for aortic arch and other complex operations, often with selective cerebral perfusion (SCP). Our previous work has demonstrated real-time evidence of improved brain protection using SCP at 18 degrees C. The purpose of this study was to evaluate the utility of SCP at warmer temperatures (25 degrees C) and its impact on operating times. METHODS Piglets undergoing cardiopulmonary bypass (CPB) and 60 min of HCA were assigned to three groups: 18 degrees C without SCP, 18 degrees C with SCP and 25 degrees C with SCP (n=8 animals per group). CPB flows were 100 ml kg(-1) min(-1) using pH-stat management. SCP flows were 10 ml kg(-1) min(-1) via the innominate artery. Cerebral oxygenation was monitored using NIRS (near-infrared spectroscopy). A microdialysis probe placed into the cerebral cortex had samples collected every 15 min. Animals were recovered for 4h after separation from CPB. All data are presented as mean+/-standard deviation (SD; p<0.05, significant). RESULTS Cerebral oxygenation was preserved during deep and tepid HCA with SCP, in contrast to deep HCA without SCP (p<0.05). Deep HCA at 18 degrees C without SCP resulted in significantly elevated brain lactate (p<0.01) and glycerol (p<0.01), while the energy substrates glucose (p<0.001) and pyruvate (p<0.001) were significantly depleted. These derangements were prevented with SCP at 18 degrees C and 25 degrees C. The lactate/pyruvate ratio (L/P) was profoundly elevated following HCA alone (p<0.001) and remained persistently elevated throughout recovery (p<0.05). Piglets given SCP during HCA at 18 degrees C and 25 degrees C maintained baseline L/P ratios. Mean operating times were significantly shorter in the 25 degrees C group compared to both 18 degrees C groups (p<0.05) without evidence of significant acidemia. CONCLUSION HCA results in cerebral hypoxia, energy depletion and ischaemic injury, which are attenuated with the use of SCP at both 18 degrees C and 25 degrees C. Procedures performed at 25 degrees C had significantly shorter operating times while preserving end organs.

Assessing transneuronal dysfunction utilizing manganese‐enhanced MRI (MEMRI)

In this study we utilized manganese‐enhanced MRI (MEMRI) to evaluate the in vivo transneuronal efficiency of manganese ion (Mn2+) movement as a means to assess overall changes in neuronal function. We designated this extension the manganese transfer index (MTI) value. To evaluate the MTI value as an index of transneuronal physiology we examined both pharmacological agents and different mouse models of neuronal dysfunction. We found that treatment with isoflurane, which attenuates synaptic vesicle release, or memantine, which attenuates postsynaptic uptake of Ca2+ as well as Mn2+, resulted in a decrease in the MTI value. Furthermore, we evaluated if changes in the MTI value can be detected in three knockout mice with altered brain function accompanied either with or without neurodegeneration. Our data demonstrate that the MTI values either decreased or increased in response to different functional as well as anatomical changes. These results demonstrate the potential utility of the MTI value as an in vivo index for the detection of changes in neuronal function in animal models of human disease. Magn Reson Med 60:169–175, 2008.

NADPH oxidase mediates β-amyloid peptide-induced activation of ERK in hippocampal organotypic cultures

BackgroundPrevious studies have shown that beta amyloid (Aβ) peptide triggers the activation of several signal transduction cascades in the hippocampus, including the extracellular signal-regulated kinase (ERK) cascade. In this study we sought to characterize the cellular localization of phosphorylated, active ERK in organotypic hippocampal cultures after acute exposure to either Aβ (1-42) or nicotine.ResultsWe observed that Aβ and nicotine increased the levels of active ERK in distinct cellular localizations. We also examined whether phospho-ERK was regulated by redox signaling mechanisms and found that increases in active ERK induced by Aβ and nicotine were blocked by inhibitors of NADPH oxidase.ConclusionOur findings indicate that NADPH oxidase-dependent redox signaling is required for Aβ-induced activation of ERK, and suggest a similar mechanism may occur during early stages of Alzheimers disease.

Selective cerebral perfusion: real-time evidence of brain oxygen and energy metabolism preservation.

BACKGROUND Deep hypothermic circulatory arrest (DHCA) is commonly used for complex cardiac operations in children, often with selective cerebral perfusion (SCP). Little data exist concerning the real-time effects of DHCA with or without SCP on cerebral metabolism. Our objective was to better define these effects, focusing on brain oxygenation and energy metabolism. METHODS Piglets undergoing cardiopulmonary bypass were assigned to either 60 minutes of DHCA at 18 degrees C (n = 9) or DHCA with SCP at 18 degrees C (n = 8), using pH-stat management. SCP was administered at 10 mL/kg/min. A cerebral microdialysis catheter was implanted into the cortex for monitoring of cellular ischemia and energy stores. Cerebral oxygen tension and intracranial pressure also were monitored. After DHCA with or without SCP, animals were recovered for 4 hours off cardiopulmonary bypass. RESULTS With SCP, brain oxygen tension was preserved in contrast to DHCA alone (p < 0.01). Deep hypothermic circulatory arrest was associated with marked elevations of lactate (p < 0.01), glycerol (p < 0.01), and the lactate to pyruvate ratio (p < 0.001), as well as profound depletion of the energy substrates glucose (p < 0.001) and pyruvate (p < 0.001). These changes persisted well into recovery. With SCP, no significant cerebral microdialysis changes were observed. A strong correlation was demonstrated between cerebral oxygen levels and cerebral microdialysis markers (p < 0.001). CONCLUSIONS Selective cerebral perfusion preserves cerebral oxygenation and attenuates derangements in cerebral metabolism associated with DHCA. Cerebral microdialysis provides real-time metabolic feedback that correlates with changes in brain tissue oxygenation. This model enables further study and refinement of strategies aiming to limit brain injury in children requiring complex cardiac operations.

In Vitro and In Vivo Magnetic Resonance Imaging (MRI) Detection of GFP through Magnetization Transfer Contrast (MTC)

Green fluorescent protein (GFP) is a widely utilized molecular marker of gene expression. However, its use in in vivo imaging has been restricted to transparent tissue mainly due to the tissue penetrance limitation of optical imaging. Here, we report a novel approach to detect GFP with Magnetization transfer contrast (MTC) magnetic resonance imaging (MRI). MTC is an MRI methodology currently utilized to detect macromolecule changes such as decrease in myelin and increase in collagen content. We employed MTC MRI imaging to detect GFP both in vitro and in in vivo mouse models. We demonstrated that our approach produces values that are protein specific, and concentration dependent. This approach provides a flexible, non-invasive in vivo molecular MRI imaging strategy that is dependent upon the presence and concentration of the GFP reporter.