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ABSTRACT: Methamphetamine increases the release and blocks the reuptake of dopamine. The moderate activation of dopamine receptors may elicit neuroprotective effects. We have recently demonstrated that low doses of methamphetamine reduce neuronal loss after ischemic injury. On the basis of this finding, we hypothesized that methamphetamine could also prevent neuronal loss and improve functional behavior after severe traumatic brain injury (TBI).
The rat lateral fluid percussion injury model was used to generate severe TBI. Three hours after injury, animals were treated with saline or methamphetamine. Neurological severity scores and foot fault assessments were used to determine whether treatment enhanced recovery after injury. The potential for methamphetamine treatment to improve cognitive function was assessed using the Morris water maze. Forty-eight hours after injury, paraffin-embedded brain sections were TUNEL stained to measure apoptotic cell death. Sections were also stained with antibody to doublecortin to quantify immature neurons within the dentate gyrus.
Treatment with low-dose methamphetamine significantly reduced both behavioral and cognitive dysfunction after severe TBI. Methamphetamine-treated animals scored significantly lower on neurological severity scores and had significantly less foot faults after TBI compared with saline-treated control rats. Furthermore, methamphetamine treatment restored learning and memory function to near normal ability after TBI. At 48 hours after injury, apoptotic cell death within the hippocampus was significantly reduced, and the presence of immature neurons was significantly increased in methamphetamine-treated rats compared with saline-treated controls.
Treatment with low-dose methamphetamine after severe TBI elicits a robust neuroprotective response resulting in significant improvements in behavioral and cognitive functions.
The journal of trauma and acute care surgery. 08/2012; 73(2 Suppl 1):S165-72.
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ABSTRACT: Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.
Free radical biology & medicine 06/2012; 53(5):1139-51. · 5.42 Impact Factor
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Thomas F Rau,
Qing Lu,
Shruti Sharma,
Xutong Sun,
Gregory Leary,
Matthew L Beckman,
Yali Hou,
Mark S Wainwright,
Michael Kavanaugh, David J Poulsen,
Stephen M Black
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ABSTRACT: Mitochondrial dysfunction characterized by depolarization of mitochondrial membranes and the initiation of mitochondrial-mediated apoptosis are pathological responses to hypoxia-ischemia (HI) in the neonatal brain. Carnitine metabolism directly supports mitochondrial metabolism by shuttling long chain fatty acids across the inner mitochondrial membrane for beta-oxidation. Our previous studies have shown that HI disrupts carnitine homeostasis in neonatal rats and that L-carnitine can be neuroprotective. Thus, this study was undertaken to elucidate the molecular mechanisms by which HI alters carnitine metabolism and to begin to elucidate the mechanism underlying the neuroprotective effect of L-carnitine (LCAR) supplementation. Utilizing neonatal rat hippocampal slice cultures we found that oxygen glucose deprivation (OGD) decreased the levels of free carnitines (FC) and increased the acylcarnitine (AC): FC ratio. These changes in carnitine homeostasis correlated with decreases in the protein levels of carnitine palmitoyl transferase (CPT) 1 and 2. LCAR supplementation prevented the decrease in CPT1 and CPT2, enhanced both FC and the AC∶FC ratio and increased slice culture metabolic viability, the mitochondrial membrane potential prior to OGD and prevented the subsequent loss of neurons during later stages of reperfusion through a reduction in apoptotic cell death. Finally, we found that LCAR supplementation preserved the structural integrity and synaptic transmission within the hippocampus after OGD. Thus, we conclude that LCAR supplementation preserves the key enzymes responsible for maintaining carnitine homeostasis and preserves both cell viability and synaptic transmission after OGD.
PLoS ONE 01/2012; 7(9):e40881. · 4.09 Impact Factor
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ABSTRACT: The pathological basis of neonatal hypoxia-ischemia (HI) brain damage is characterized by neuronal cell loss. Oxidative stress is thought to be one of the main causes of HI-induced neuronal cell death. The p38 mitogen-activated protein kinase (MAPK) is activated under conditions of cell stress. However, its pathogenic role in regulating the oxidative stress associated with HI injury in the brain is not well understood. Thus, this study was conducted to examine the role of p38 MAPK signaling in neonatal HI brain injury using neonatal rat hippocampal slice cultures exposed to oxygen/glucose deprivation (OGD). Our results indicate that OGD led to a transient increase in p38 MAPK activation that preceded increases in superoxide generation and neuronal death. This increase in neuronal cell death correlated with an increase in the activation of caspase-3 and the appearance of apoptotic neuronal cells. Pre-treatment of slice cultures with the p38 MAPK inhibitor, SB203580, or the expression of an antisense p38 MAPK construct only in neuronal cells, through a Synapsin I-1-driven adeno-associated virus vector, inhibited p38 MAPK activity and exerted a neuroprotective effect as demonstrated by decreases in OGD-mediated oxidative stress, caspase activation and neuronal cell death. Thus, we conclude that the activation of p38 MAPK in neuronal cells plays a key role in the oxidative stress and neuronal cell death associated with OGD.
European Journal of Neuroscience 09/2011; 34(7):1093-101. · 3.63 Impact Factor
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ABSTRACT: High doses of methamphetamine induce the excessive release of dopamine resulting in neurotoxicity. However, moderate activation of dopamine receptors can promote neuroprotection. Therefore, we used in vitro and in vivo models of stroke to test the hypothesis that low doses of methamphetamine could induce neuroprotection. We demonstrate that methamphetamine does induce a robust, dose-dependent, neuroprotective response in rat organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD). A similar dose dependant neuroprotective effect was observed in rats that received an embolic middle cerebral artery occlusion (MCAO). Significant improvements in behavioral outcomes were observed in rats when methamphetamine administration delayed for up to 12 h after MCAO. Methamphetamine-mediated neuroprotection was significantly reduced in slice cultures by the addition of D1 and D2 dopamine receptor antagonist. Treatment of slice cultures with methamphetamine resulted in the dopamine-mediated activation of AKT in a PI3K dependant manner. A similar increase in phosphorylated AKT was observed in the striatum, cortex and hippocampus of methamphetamine treated rats following MCAO. Methamphetamine-mediated neuroprotection was lost in rats when PI3K activity was blocked by wortmannin. Finally, methamphetamine treatment decreased both cleaved caspase 3 levels in slice cultures following OGD and TUNEL staining within the striatum and cortex in rats following transient MCAO. These data indicate that methamphetamine can mediate neuroprotection through activation of a dopamine/PI3K/AKT-signaling pathway.
Neuropharmacology 05/2011; 61(4):677-86. · 4.81 Impact Factor
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ABSTRACT: The lack of effective treatments for many forms of hearing and vestibular disorders has produced interest in virally mediated gene therapies. However, to develop a gene therapy strategy that would successfully treat inner ear disorders, appropriate viral vectors capable of transfecting cochlear and support cells must be identified. While virally mediated gene transfer into the inner ear has been accomplished using herpes simplex type I virus, vaccinia virus, retroviruses, adenovirus, and adeno-associated virus (AAV), we will restrict our discussion to AAV and adenoviral vectors. Issues such as vector toxicity and load, viral serotype and backbone, and promoter specificity are discussed and contrasted for both in vivo vs. in vitro inner ear gene transfer.
Advances in oto-rhino-laryngology 02/2009; 66:87-98.
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C Sean Esslinger,
Shailesh Agarwal,
John Gerdes,
Paul A Wilson,
Erin S Davis,
Alicia N Awes,
Erin O'Brien,
Teri Mavencamp,
Hans P Koch, David J Poulsen,
Joseph F Rhoderick,
A Richard Chamberlin,
Michael P Kavanaugh,
Richard J Bridges
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ABSTRACT: The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, especially related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacology and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of beta-substituted aspartate analogues identify L-beta-benzyl-aspartate (L-beta-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic analysis of D-[(3)H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-beta-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes identify L-beta-BA as a non-substrate inhibitor. Analyzing L-beta-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: (1) a highly conserved positioning of the electrostatic carboxyl and amino groups; (2) nearby regions that accommodate select structural modifications (cyclopropyl rings, methyl groups, oxygen atoms); and (3) a unique region L-beta-threo-BA occupied by the benzyl moieties of L-TBOA, L-beta-threo-BA and related analogues. It is plausible that the preference of L-beta-threo-BA and L-TBOA for EAAT3 and EAAT2, respectively, could reside in the latter two pharmacophore regions.
Neuropharmacology 12/2005; 49(6):850-61. · 4.81 Impact Factor
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ABSTRACT: More than 28 million Americans suffer from various forms of hearing loss. The lack of effective treatments for many forms of hearing disorders has prompted interest in the potential application of gene delivery techniques to treat both inherited and pathological hearing disorders. However, to develop a gene therapy strategy that will successfully treat hearing disorders, appropriate vectors that are capable of transducing cochlear hair cells and support cells must be identified. In the present study, we examined the efficiency with which AAV vectors (serotypes 1, 2, and 5) transduce hair cells and support cells in cochlear explants from P0 and E13 mice. We further examined the ability of the CBA and GFAP promoters to drive expression of a GFP marker gene in hair cells and support cells. Robust GFP expression was observed in hair cells and support cells following transduction of primary murine cochlear explants with AAV serotypes 1 and 2, but not serotype 5. The CBA promoter predominantly drove GFP expression in hair cells. In contrast, strong expression from the GFAP promoter was observed primarily in support cells. Thus, using AAV vectors and specific promoters, cell-type-specific expression of transgenes can be established within the cochlea.
Molecular Therapy 07/2005; 11(6):843-8. · 6.87 Impact Factor
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ABSTRACT: Excitatory amino acid transporters (EAATs) maintain the balance between pathological and physiological conditions by limiting the extracellular concentration of glutamate within the CNS and thus preventing excitotoxic injury. The loss of EAAT2 has been associated with the development of neurological diseases such as amyotrophic lateral sclerosis. It has therefore been suggested that the over-expression of specific EAATs may provide some degree of neuroprotection. However, the inability to isolate and study the function of the different EAAT isoforms in a cell type-specific manner has made it difficult to determine the exact contribution of individual EAATs toward neuroprotection or neurodegeneration in the context of excitotoxic injury. To address this question, we transduced hippocampal slice cultures from 1-week-old C57B/6 mice with recombinant adeno-associated virus carrying an EAAT2 gene expression cassette. EAAT2 gene expression was driven in neurons with the neuron-specific enolase promoter. Using this model system, we were able to induce a significant increase in the expression of functional EAAT2. Consequently, a significant increase in CA1 neuronal damage was observed in slices over-expressing EAAT2 in neurons following an acute exposure to exogenous glutamate. These data suggest that the increased expression of EAAT2 within neurons may contribute to neurodegeneration.
European Journal of Neuroscience 05/2005; 21(8):2291-6. · 3.63 Impact Factor
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ABSTRACT: Descriptive histologic analysis of spinal cord gene therapy.
To maximize protein expression in rat spinal cord using recombinant adenoassociate virus viral vector.
There are few reports of spinal cord genetic transfer. There have been no reports that compare techniques to increase protein expression through genetic alterations or have illustrated successful genetic transfer to spinal cord astrocytes.
Adenoassociate virus constructs were packaged using three separate plasmids: a cis plasmid with the expression cassettes (pAM/neuron-specific enolase/green fluorescent protein/woodchuck posttranscriptional regulatory element/simian virus 40/polyadenylase or pAM/glial fibrillary acid protein/green fluorescent protein/woodchuck posttranscriptional regulatory element/simian virus 40/polyadenylase), the Ad-adenoassociate virus helper trans plasmid, and the essential region from the adenovirus genome (pFDelta6). The adenoassociate virus 2/5 capsid gene replaces the adenoassociate virus 2 capsid region in the trans construct, resulting in a different cellular tropism. Thirty-two adult (300-375 g) male Sprague-Dawley rats underwent L1 laminectomies. A total volume of 6 microL was injected directly into the spinal cord parenchyma at a rate of 600 nL/min with adenoassociate virus 2/glial fibrillary acid protein/green fluorescent protein, adenoassociate virus 2/neuron-specific enolase/green fluorescent protein, adenoassociate virus 2/5/glial fibrillary acid protein/green fluorescent protein, or adenoassociate virus 2/5/neuron-specific enolase/green fluorescent protein and either a low- (4 x 10(8)) or high-titer (1 x 10(10)) viral solution.
The gene expression (green fluorescent protein reporter) was present in the cell bodies and axonal processes of all adenoassociate virus/green fluorescent protein constructs. However, a greater spread of virus was observed in rats injected with adenoassociate virus 2/5 compared with adenoassociate virus 2. In addition, more neurons were transduced with adenoassociate virus 2/5 than adenoassociate virus 2, and green fluorescent protein expression in neurons transduced with adenoassociate virus 2/5 appeared more intense compared with adenoassociate virus 2 neurons. The difference observed between adenoassociate virus 2 and adenoassociate virus 2/5 at 4 x 10(8) genomic particles/mL was not as profound when the virus titer was raised to 1 x 10(10) genomic particles/mL. Green fluorescent protein expression was observed in astrocytes following injection of rat spinal cords with either adenoassociate virus 2 or adenoassociate virus 2/5 carrying the glial fibrillary acid protein/green fluorescent protein construct. However, unlike neuron-specific enolase-driven expression, there was less overall expression, but a substantial increase in green fluorescent protein expression was observed with adenoassociate virus 2/5 compared with adenoassociate virus 2 with high virus titers. Furthermore, unlike the neuron-specific enolase promoter, glial fibrillary acid protein-driven expression of green fluorescent protein was not restricted to astrocytes alone. The glial fibrillary acid protein construct was able to transfect glial cells and maintain glial expression.
Adenoassociate virus can readily transduce spinal cord neurons and is an efficient nonpathologic vector to deliver expression cassettes. Increased titers and the adenoassociate virus 2/5 serotype appeared to maximize expression.
Spine 01/2005; 29(24):2787-92. · 2.08 Impact Factor
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ABSTRACT: An incomplete understanding of the pathological processes involved in neurodegeneration and dysfunction of spinal cord injuries and diseases makes these disorders difficult to treat. Repair of damaged or genetically impaired spinal cord also has been limited by the complexity, cellular heterogeneity, and relative inaccessibility of the tissue. Thus, therapeutic options for the treatment of either chronic spinal cord diseases such as amyotrophic lateral sclerosis or acute spinal cord injuries have been rather limited. Potential new therapeutic targets are being identified as our understanding of the molecular pathology involved in neural injury and regeneration increases. Recent advances in gene transfer techniques have made gene therapy a more realistic and viable strategy for the treatment of a broad range of spinal cord disorders. This review summarizes the current state of knowledge regarding the limitations and recent advances in gene therapy and potential application of this technology toward spinal cord injury and disease.
The journal of spinal cord medicine 02/2002; 25(1):2-9. · 2.11 Impact Factor
