Thomas F. Rau

Selective overexpression of excitatory amino acid transporter 2 (EAAT2) in astrocytes enhances neuroprotection from moderate but not severe hypoxia–ischemia

Attempts have been made to elevate excitatory amino acid transporter 2 (EAAT2) expression in an effort to compensate for loss of function and expression associated with disease or pathology. Increased EAAT2 expression has been noted following treatment with beta-lactam antibiotics, and during ischemic preconditioning (IPC). However, both of these conditions induce multiple changes in addition to alterations in EAAT2 expression that could potentially contribute to neuroprotection. Therefore, the aim of this study was to selectively overexpress EAAT2 in astrocytes and characterize the cell type specific contribution of this transporter to neuroprotection. To accomplish this we used a recombinant adeno-associated virus vector, AAV1-glial fibrillary acidic protein (GFAP)-EAAT2, designed to selectively drive the overexpression of EAAT2 within astrocytes. Both viral-mediated gene delivery and beta-lactam antibiotic (penicillin-G) treatment of rat hippocampal slice cultures resulted in a significant increase in both the expression of EAAT2, and dihydrokainate (DHK) sensitive glutamate uptake. Penicillin-G provided significant neuroprotection in rat hippocampal slice cultures under conditions of both moderate and severe oxygen glucose deprivation (OGD). In contrast, viral-mediated overexpression of EAAT2 in astrocytes provided enhanced neuroprotection only following a moderate OGD insult. These results indicate that functional EAAT2 can be selectively overexpressed in astrocytes, leading to enhanced neuroprotection. However, this cell type specific increase in EAAT2 expression offers only limited protection compared to treatment with penicillin-G.

Treatment with low-dose methamphetamine improves behavioral and cognitive function after severe traumatic brain injury

BACKGROUND 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). METHODS 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. RESULTS 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. CONCLUSION Treatment with low-dose methamphetamine after severe TBI elicits a robust neuroprotective response resulting in significant improvements in behavioral and cognitive functions.

Low dose methamphetamine mediates neuroprotection through a PI3K-AKT pathway

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.

Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures.

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.

Increased p38 mitogen‐activated protein kinase signaling is involved in the oxidative stress associated with oxygen and glucose deprivation in neonatal hippocampal slice cultures

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.

Oxygen Glucose Deprivation in Rat Hippocampal Slice Cultures Results in Alterations in Carnitine Homeostasis and Mitochondrial Dysfunction

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.

Administration of low dose methamphetamine 12 h after a severe traumatic brain injury prevents neurological dysfunction and cognitive impairment in rats

We recently published data that showed low dose of methamphetamine is neuroprotective when delivered 3 h after a severe traumatic brain injury (TBI). In the current study, we further characterized the neuroprotective potential of methamphetamine by determining the lowest effective dose, maximum therapeutic window, pharmacokinetic profile and gene expression changes associated with treatment. Graded doses of methamphetamine were administered to rats beginning 8 h after severe TBI. We assessed neuroprotection based on neurological severity scores, foot fault assessments, cognitive performance in the Morris water maze, and histopathology. We defined 0.250 mg/kg/h as the lowest effective dose and treatment at 12 h as the therapeutic window following severe TBI. We examined gene expression changes following TBI and methamphetamine treatment to further define the potential molecular mechanisms of neuroprotection and determined that methamphetamine significantly reduced the expression of key pro-inflammatory signals. Pharmacokinetic analysis revealed that a 24-hour intravenous infusion of methamphetamine at a dose of 0.500 mg/kg/h produced a plasma Cmax value of 25.9 ng/ml and a total exposure of 544 ng/ml over a 32 hour time frame. This represents almost half the 24-hour total exposure predicted for a daily oral dose of 25mg in a 70 kg adult human. Thus, we have demonstrated that methamphetamine is neuroprotective when delivered up to 12 h after injury at doses that are compatible with current FDA approved levels.

Plasma micro-RNA biomarkers for diagnosis and prognosis after traumatic brain injury: A pilot study.

Prediction of post-concussive syndrome after apparent mild traumatic brain injury (TBI) and subsequent cognitive recovery remains challenging, with substantial limitations of current methods of cognitive testing. This pilot study aimed to determine if levels of micro ribonucleic acids (RNAs) circulating in plasma are altered following TBI, and if changes to levels of such biomarkers over time could assist in determination of prognosis after TBI. Patients were enrolled after TBI on presentation to the Emergency Department and allocated to three groups: A - TBI (physical trauma to the head), witnessed loss of consciousness, amnesia, GCS=15, a normal CT Brain and a recorded first pass after post-traumatic amnesia (PTA) scale; B TBI, witnessed LOC, amnesia, GCS=15, a normal CT brain and a PTA scale test fail and: C - TBI and initial GCS <13 on arrival to the ED. Venous blood was collected at three time points (arrival, day 5 and day 30). Isolation of cell-free total RNA was then assayed using a custom miRNA PCR array. Two micro-RNAs, mir142-3p and mir423-3p demonstrated potential clinical utility differentiating patients after mild head injury into those at greater risk of developing amnesia and therefore, post-concussive syndromes. In addition, these miRNA demonstrated a decrease in expression over time, possibly indicative of brain healing after the injury. Further evaluation of these identified miRNA markers with larger patient cohorts, correlation with clinical symptoms and analysis over longer time periods are essential next steps in developing objective markers of severity of TBI.

The neuroprotective potential of low-dose methamphetamine in preclinical models of stroke and traumatic brain injury

Methamphetamine is a psychostimulant that was initially synthesized in 1920. Since then it has been used to treat attention deficit hyperactive disorder (ADHD), obesity and narcolepsy. However, methamphetamine has also become a major drug of abuse worldwide. Under conditions of abuse, which involve the administration of high repetitive doses, methamphetamine can produce considerable neurotoxic effects. However, recent evidence from our laboratory indicates that low doses of methamphetamine can produce robust neuroprotection when administered within 12h after severe traumatic brain injury (TBI) in rodents. Thus, it appears that methamphetamine under certain circumstances and correct dosing can produce a neuroprotective effect. This review addresses the neuroprotective potential of methamphetamine and focuses on the potential beneficial application for TBI.

The effects of mild traumatic brain injury on postural control

ABSTRACT Primary objective: The purpose of this study was to investigate the effects of mild traumatic brain injury (mTBI) on multiple postural indices that characterize body sway behaviour. Methods and procedures: The body’s centre of pressure (COP) displacement was recorded from 11 individuals with a history of mTBI (29.4 ± 6.7 years old) and 11 healthy controls (26.8 ± 3.7 years old) performing bipedal stance on a force platform for 120 seconds. Spatio-temporal (area, amplitude and mean velocity of the COP displacement) and frequency characteristics (frequency containing 80% of the power spectral density) of the body oscillation, as well as its dynamic characteristics (sample entropy estimate of the COP displacement) were extracted from COP signals. Main outcomes and results: All postural indices studied were significantly affected by mTBI (p < 0.010). Participants with a history of mTBI presented a larger, slower, and more random body oscillation compared to controls. Conclusion: The results suggest that (a) balance deficits can be recognized as an effect of mTBI; (b) balance deficits induced by mTBI are multi-dimensional, affecting all three domains included in this study; and (c) the postural indices employed in this study are potential markers to detect changes in postural control following mTBI.