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The number of putative neuroprotective compounds with antioxidant activity described in the literature continues to grow. Although these compounds are validated using a variety of in vivo and in vitro techniques, they are often evaluated initially using in vitro cell culture techniques in order to establish toxicity and effective concentrations. Both in vivo and in vitro methodologies have their respective advantages and disadvantages, including, but not limited to, cost, time, use of resources and technical limitations. This review expands on the inherent benefits and drawbacks of in vitro and in vivo methods for assessing neuroprotection, especially in light of proper evaluation of compound efficacy and neural bioavailability. For example, in vivo studies can better evaluate the effects of protective compounds and/or its metabolites on various tissues, including the brain, in the whole animal, whereas in vitro studies can better discern the cellular and/or mechanistic effects of compounds. In particular, we aim to address the question of appropriate and accurate extrapolation of findings from in vitro experiment-where compounds are often directly applied to cellular extracts, potentially at higher concentrations than would ever cross the blood-brain barrier—to the more complex scenario of neuroprotection due to pharmacodynamics in vivo.

Oxyresveratrol is a potent antioxidant and free-radical scavenger found in mulberry wood (Morus alba L.) with demonstrated protective effects against cerebral ischemia. We analyzed the neuroprotective ability of oxyresveratrol using an in vitro model of stretch-induced trauma in co-cultures of neurons and glia, or by exposing cultures to high levels of glutamate. Cultures were treated with 25 mu M, 50 mu M or 100 mu M oxyresveratrol at the time of injury. Trauma produced marked neuronal death when measured 24 h post-injury, and oxyresveratrol significantly inhibited this death. Microscopic examination of glia suggested signs of toxicity in cultures treated with 100 mu M oxyresveratrol, as demonstrated by elevated S-100B protein release and a high proportion of cells with condensed nuclei. Cultures exposed to glutamate (100 mu M) for 24 h exhibited similar to 37% neuronal loss, which was not inhibited by oxyresveratrol. These results show that the two pathologies of high glutamate exposure and trauma are differentially affected by oxyresveratrol treatment in vitro. Further studies using oxyresveratrol in trauma models are warranted, as toxicity to glia could be beneficial by inhibiting reactive gliosis, which often occurs after trauma. Crown Copyright (C) 2012 Published by Elsevier B. V. All rights reserved.

Traumatic brain injury (TBI) consists of two phases: an immediate phase in which damage is caused as a direct result of the mechanical impact; and a late phase of altered biochemical events that results in delayed tissue damage and is therefore amenable to therapeutic treatment. Because the molecular mechanisms of delayed post-traumatic neuronal cell death are still poorly understood, we investigated whether apoptosis-inducing factor (AIF), a pro-apoptotic mitochondrial molecule and the key factor in the caspase-independent, cell death signaling pathway, plays a causal role in neuronal death following TBI. Using an in vitro model of neuronal stretch injury, we demonstrated that AIF translocated from mitochondria to the nucleus of neurons displaying axonal disruption, chromatin condensation, and nuclear pyknosis in a caspase-independent manner, whereas astrocytes remained unaffected. Similar findings were observed following experimental TBI in mice, where AIF translocation to the nucleus coincided with delayed neuronal cell death in both cortical and hippocampal neurons. Down-regulation of AIF in vitro by siRNA significantly reduced stretch-induced neuronal cell death by 67%, a finding corroborated in vivo using AIF-deficient harlequin mutant mice, where secondary contusion expansion was significantly reduced by 44%. Hence, our current findings demonstrate that caspase-independent, AIF-mediated signaling pathways significantly contribute to post-traumatic neuronal cell death and may therefore represent novel therapeutic targets for the treatment of TBI.

The cerebellum has been shown to be vulnerable to global ischemic damage in tightly controlled zones of Purkinje cells (PCs) that lack aldolase C, an enzyme critical for glycolysis. Here, we investigated whether aldolase C-negative PCs were more likely to die after cerebral trauma in vivo, and whether this death was mediated by excitotoxic [alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated] means in vitro. Mice were subjected to controlled cortical impact, or remained uninjured, and were killed at 6 h, 24 h or 7 days after injury. Cerebellar sections (both ipsilateral and contralateral to the site of cerebral injury) were stained against aldolase C and calbindin (a marker of PCs). The number of viable, calbindin-positive PCs decreased significantly at 24 h and 7 days after injury, and the percentage of surviving, aldolase C-positive PCs significantly increased at those time-points. In addition, we subjected murine cerebellar cultures to AMPA (30 microm, 20 min), which killed a significant number of PCs at 24 h post-treatment. A similar number of PCs was lost after transfection with aldolase C siRNA, and this effect was exacerbated in transfected cultures treated with AMPA. The results from the present study indicate that aldolase C provides marked neuroprotection to PCs after trauma and excitotoxicity.

The homeostasis of intracellular Cl(-) concentration ([Cl(-)](i)) is critical for neuronal function, including gamma-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl(-)](i) using a transgenetically expressed Cl(-)-sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 microm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl(-)-free solution, demonstrating that the EYFP signal represented an increase in [Cl(-)](i). Similar to glutamate, a rise in [Cl(-)](i) was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K(+)], indicating that an increase in driving force for Cl(-) suffices to increase [Cl(-)](i). To elucidate the membrane mechanisms mediating the Cl(-) influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl(-)](i) was resistant to furosemide, bumetanide and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl(-)](i) via several pathways involving NFA- and NPPB-sensitive anion channels and GABA(A) receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl(-) transporters. The present study highlights the vulnerability of [Cl(-)](i) homeostasis after membrane depolarization in neurons.

Phencyclidine (PCP) produces psychotomimetic effects in humans that resemble schizophrenia symptoms. In an effort to screen compounds for antipsychotic activity, preclinical researchers have investigated whether these compounds block PCP-induced behaviors in animals. In the present study, the atypical antipsychotic clozapine was tested in combination with an active dose of PCP in two-lever drug discrimination and mixed signalled-unsignalled differential-reinforcement-of-low-rates (DRL) procedures. PCP produced distinctive effects in each task: it substituted for the training dose in PCP discrimination and it increased the number of responses with short (

__Einleitung__ Das leichte Schadel-Hirn-Trauma (leichtes SHT, engl. „minor traumatic brain injury, mTBI”) wird im Sport oft bagatellisiert. Definiert ist das leichte SHT hauptsachlich als eine klinisch transiente globale Hirnfunktionsstorung durch aussere Krafteinwirkung unter Abwesenheit von fokalen neurologischen Ausfallen und spezifischen neurologischen Befunden. Die wesentlichen Merkmale sind Bewusstseinsverlust fur maximal 20-30 Minuten, ein Glasgow Coma Scale (GCS) Score von 13-15, keine intrakranialen Abnormalitaten in der Computertomographie des Schadels, sowie Verwirrung und/oder Amnesie fur weniger als 24 Stunden. Der behandelnde Arzt geht meist davon aus, dass es sich hierbei um eine nicht-strukturelle Hirnverletzung handelt, die in jedem Fall eine blande Prognose hat. Im folgenden Artikel wird aufgezeigt, dass es sich auch beim leichten Schadel-Hirn-Trauma um eine behandlungsbedurftige strukturelle Hirnschadigung mit potentiell akut lebensbedrohlichen Folgen handelt. __Methodik__ Dieser Kurzbericht basiert auf dem „First International Meeting on Minor Traumatic Brain Injury” vom 28. Februar bis 4. Marz 2005 in Samedan/Schweiz unter der Leitung von Dr. Biasca. __Ergebnisse__ Das leichte SHT ist eine Verletzungsform, die bei allen Sportarten, bei welchen Geschwindigkeit und Kraft zusammenwirken, insbesondere durch Beschleunigung mit abrupten Rotationsbewegungen vorkommen kann. Die typischen sZeichen sind Verwirrtheit und Amnesie. Der Bewusstseinsverlust ist nicht obligatorisch. Warnsymptome sind Kopfschmerz, Schwindel oder Ubelkeit. Der Schweregrad eines leichten SHTs kann klinisch am besten anhand dem Auftreten und der Lange einer Amnesie beurteilt werden. Jegliche Form einer retrograden Amnesie ist mit einem 10-fach hoheren Risiko eines schlechteren Krankeitsverlaufs assoziiert. Dagegen scheint kein Pradilektionswert fur den Bewusstseinsverlust im Hinblick auf die Prognose nach einer mTBI vorzuliegen.

Beneficial health effects of cranberries (CBs) and wild blueberries (BBs), such as reduced levels of oxidative stress, have been demonstrated in feeding studies. These Vaccinium berries contain high levels of flavonoids; however, the bioavailability of flavonoids is generally low. We investigated the in vitro effects of these berries on intestinal cells, focusing on mitigating oxidative stress and associated reactive oxygen species (ROS). First, we simulated the passage of CB and BB through the gastrointestinal (GI) tract by treating berry homogenates to a battery of digestive enzymes. Then, Caco-2 cells, a model of small intestine epithelial uptake, were exposed to these homogenates for 60 min. Using a cell-free assay, we found that the antioxidant activity in CB homogenates was not affected by these enzymes, but that BB homogenates treated with gut enzymes had 43% lower free-radical quenching activity (P0.05). However, both of the enzyme-treated homogenates were still able to counteract the ROS-generating ability of H2O2 added exogenously to Caco-2 cells. Berry homogenates also increased mitochondrial metabolic rates at 60 min posttreatment, as measured by MTT assays. Enzyme-treated CB (but not BB) homogenates increased the levels of reduced glutathione (GSH) relative to oxidized glutathione (GSSG), a critical indicator of the cellular redox state (P0.05). Our data suggest that CBs do not lose their antioxidant ability when passing through the GI tract, and specifically, digested CB may serve to enhance cytoprotective effects in intestinal cells by reducing potential damage caused by free radicals and ROS derived from other food sources.

The spontaneously hypertensive stroke-prone (SHRSP) rat is a commonly used model of cerebrovascular disease and hypertension. SHRSP rats have been shown to develop stroke-related symptoms (SRS) by age 14 weeks when fed a purified diet, such as AIN-93G, supplemented with 1% NaCl. We conducted a pathology pilot study to compare the incidence of SRS in SHRSP rats fed either AIN-93G (with 1% NaCl in drinking water) or commercially available rat chow (with 4% NaCl in the diet), starting at 8 weeks of age. These results prompted us to analyze data from 5 earlier feeding trials using SHRSP rats. Overall, we found that SHRSP rats fed AIN-93G purified diet for 8 or 17 weeks did not demonstrate SRS (n = 18), whereas all SHRSP rats fed lab chow exhibited SRS at age 15.1 ± 0.6 weeks (n = 23). In addition, SHRSP rats fed lab chow had decreased mass gain starting at age 13 weeks, as well as decreased feed efficiencies after the first 5 weeks of feeding (p < 0.05). In conclusion, our data suggest that diet composition is a major contributor to the onset of stroke in SHRSP rats and that diet choice should be critically evaluated based on endpoint measures in the SHRSP model.

Feeding flavonoid-rich blueberries to spontaneously hypertensive stroke-prone rats (SHRSP) lowers blood pressure. To determine whether this is due to inhibition of angiotensin-converting enzyme (ACE) activity, as seen with other flavanoid-rich foods, we fed blueberries to SHRSP and normotensive rats and analyzed ACE activity in blood and tissues. After 2 weeks on a control diet, the hypertensive rats showed 56% higher levels of ACE activity in blood as compared with the normotensive rats (p

Dementia in humans following traumatic brain injury (TBI) has been well documented in clinical populations, either after a single TBI or repeated mild TBI (rMTBI). In most cases, trauma-induced dementia follows a slow, chronic time-course, and in many cases mild injuries accumulate over time. Both single TBI and rMTBI have been modeled experimentally in vivo, most often in rodents, and various treatment strategies have been studied in order to reduce brain damage after injury. Here, we review the recent literature with regard to currently used in vivo models of TBI in animals, and studies conducted with them to investigate the link between TBI and the development of dementia-like pathology, such as that associated with Alzheimer’s disease (AD). We also discuss the potential use of in vitro models of trauma for investigating a link between trauma and dementia.

The cerebellum has been shown to be vulnerable to global ischemic damage in tightly controlled zones of Purkinje cells (PCs) that lack aldolase C, an enzyme critical for glycolysis. Here, we investigated whether aldolase C-negative PCs were more likely to die after cerebral trauma in vivo, and whether this death was mediated by excitotoxic [alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-mediated] means in vitro. Mice were subjected to controlled cortical impact, or remained uninjured, and were killed at 6 h, 24 h or 7 days after injury. Cerebellar sections (both ipsilateral and contralateral to the site of cerebral injury) were stained against aldolase C and calbindin (a marker of PCs). The number of viable, calbindin-positive PCs decreased significantly at 24 h and 7 days after injury, and the percentage of surviving, aldolase C-positive PCs significantly increased at those time-points. In addition, we subjected murine cerebellar cultures to AMPA (30 microm, 20 min), which killed a significant number of PCs at 24 h post-treatment. A similar number of PCs was lost after transfection with aldolase C siRNA, and this effect was exacerbated in transfected cultures treated with AMPA. The results from the present study indicate that aldolase C provides marked neuroprotection to PCs after trauma and excitotoxicity.

Der zweite Teil unseres Artikels zum Thema des leichten Schadel-Hirn-Traumas (minor traumatic brain injury, mTBI) im Sport stellt die neuen Erkenntnisse aus diesem Gebiet vor, basierend auf den Ergebnissen des «First International Symposium on Minor Traumatic Brain Injury», das vom 28. Februar bis zum 4. Marz 2005 in Samedan unter der Leitung von Dr. med. Erik Matser, Mitglied der Hollandischen Gesundheitsbehorde, und Dr. med. Nicola Biasca1, Medical Consultant der International Ice Hockey Federation IIHF, stattgefunden hat. Schwerpunkte sind neben der Pathophysiologie vor allem die Komplikationen durch Bagatellisierung, die Auswirkungen bei Kindern und die Verletzungspravention.

Summary Undetected brain injury in sports: minor traumatic brain injury and its consequences. Part 1 Minor traumatic brain injury (mTBI) in sport must be regarded as an injury to the brain which requires treatment. All mTBIs sustained earlier must be duly recorded and taken into consideration in drawing up a treatment plan. Therapy should follow precisely defined guidelines, e.g. the stepwise protocol of the IOC/FIFA/IIHF Concussion in Sports (CIS) Group. Sports activities should be resumed only when all physical, and also cognitive, symptoms have subsided.

Purkinje cells (PCs) present a unique cellular profile in both the cerebellum and the brain. Because they represent the only output cell of the cerebellar cortex, they play a vital role in the normal function of the cerebellum. Interestingly, PCs are highly susceptible to a variety of pathological conditions that may involve glutamate-mediated 'excitotoxicity', a term coined to describe an excessive release of glutamate, and a subsequent over-activation of excitatory amino acid (NMDA, AMPA, and kainite) receptors. Mature PCs, however, lack functional NMDA receptors, the means by which Ca(2+) enters the cell in classic hippocampal and cortical models of excitotoxicity. In PCs, glutamate predominantly mediates its effects, first via a rapid influx of Ca(2+)through voltage-gated calcium channels, caused by the depolarization of the membrane after AMPA receptor activation (and through Ca(2+)-permeable AMPA receptors themselves), and second, via a delayed release of Ca(2+) from intracellular stores. Although physiological levels of intracellular free Ca(2+) initiate vital second messenger signaling pathways in PCs, excessive Ca(2+) influx can detrimentally alter dendritic spine morphology via interactions with the neuronal cytoskeleton, and thus can perturb normal synaptic function. PCs possess various calcium-binding proteins, such as calbindin-D28K and parvalbumin, and glutamate transporters, in order to prevent glutamate from exerting deleterious effects. Bergmann glia are gaining recognition as key players in the clearance of extracellular glutamate; these cells are also high in S-100beta, a protein with both neurodegenerative and neuroprotective abilities. In this review, we discuss PC-specific mechanisms of glutamate-mediated excitotoxic cell death, the relationship between Ca(2+) and cytoskeleton, and the implications of glutamate, and S-100beta for pathological conditions, such as traumatic brain injury.

Traumatic brain injury (TBI) involves direct mechanical damage, which may be aggravated by secondary insults such as ischemia. We utilized an in vitro model of stretch-induced injury to investigate the effects of mechanical and combined mechanical/ischemic insults to cultured mouse cortical cells. Stretch injury alone caused significant neuronal loss and increased uptake of the dye, propidium iodide, suggesting cellular membrane damage to both glia and neurons. Exposure of cultures to ischemic conditions for 24 h, or a combination of stretch and 24 h of ischemia, caused greater neuronal loss compared to stretch injury alone. Next, we tested the neuroprotective effects of superoxide dismutase (SOD), and the nitric oxide (NO) synthase inhibitors 7-nitroindazole (7-NINA) and lubeluzole. In general, these agents decreased neuronal loss following stretch injury alone, but were relatively ineffective against the combined injury paradigm. A combination of SOD with 7-NINA or lubeluzole offered no additional protection than single drug treatment against stretch alone or combined injury. These results suggest that the effects of primary mechanical damage and secondary ischemia to cortical neurons are cumulative, and drugs that scavenge superoxide or reduce NO production may not be effective for treating the secondary ischemia that often accompanies TBI.

Purkinje cells (PCs) present a unique cellular profile in both the cerebellum and the brain. Because they represent the only output cell of the cerebellar cortex, they play a vital role in the normal function of the cerebellum. Interestingly, PCs are highly susceptible to a variety of pathological conditions that may involve glutamate-mediated 'excitotoxicity', a term coined to describe an excessive release of glutamate, and a subsequent over-activation of excitatory amino acid (NMDA, AMPA, and kainite) receptors. Mature PCs, however, lack functional NMDA receptors, the means by which Ca(2+) enters the cell in classic hippocampal and cortical models of excitotoxicity. In PCs, glutamate predominantly mediates its effects, first via a rapid influx of Ca(2+)through voltage-gated calcium channels, caused by the depolarization of the membrane after AMPA receptor activation (and through Ca(2+)-permeable AMPA receptors themselves), and second, via a delayed release of Ca(2+) from intracellular stores. Although physiological levels of intracellular free Ca(2+) initiate vital second messenger signaling pathways in PCs, excessive Ca(2+) influx can detrimentally alter dendritic spine morphology via interactions with the neuronal cytoskeleton, and thus can perturb normal synaptic function. PCs possess various calcium-binding proteins, such as calbindin-D28K and parvalbumin, and glutamate transporters, in order to prevent glutamate from exerting deleterious effects. Bergmann glia are gaining recognition as key players in the clearance of extracellular glutamate; these cells are also high in S-100beta, a protein with both neurodegenerative and neuroprotective abilities. In this review, we discuss PC-specific mechanisms of glutamate-mediated excitotoxic cell death, the relationship between Ca(2+) and cytoskeleton, and the implications of glutamate, and S-100beta for pathological conditions, such as traumatic brain injury.

Recent studies in vivo have shown that cells of the cerebellum, and particularly Purkinje neurons (PNs), are susceptible to damage following traumatic brain injury (TBI). To investigate more closely the effects of TBI at the cellular level, we subjected cerebellar cell cultures to injury using an in vitro model of stretch-induced mechanical trauma and found increased cell damage and neuronal loss with increasing levels of injury and time post-injury. The release of neuron-specific enolase and S-100 beta were also elevated after injury. Compared to our previous findings in hippocampal cells, S-100 beta levels were much higher in cerebellar cultures after injury, suggesting that cells from different brain regions show variable responses to mechanical trauma. Lastly, the addition of exogenous S-100 beta to uninjured cerebellar cells caused no overt change in cell viability or overall neuronal number; there were, however, fewer calbindin-positive PNs, similar to findings after stretch injury.

Summary An interesting hypothesis in the study of neurotrauma is that repeated traumatic brain injury may result in cumulative damage to cells of the brain. However, postinjury sequelae are difficult to address at the cellular level in vivo. Therefore, it is necessary to complement these studies with experiments conducted in vitro .I n this report, the effects of single and repeated traumatic injury in vitro were investigated in cultured mouse hippocampal cells using a well characterized model of stretch-induced injury. Cell damage was assessed by the level of propidium iodide (PrI) uptake and retention of fluorescein diacetate (FDA). Uninjured control wells displayed minimal PrI uptake and high levels of FDA retention. Mild, moderate and severe levels of stretch caused increasing amounts of PrI uptake, respectively, when measured at 15 min and 24 h post-injury, indicating increased cellular damage with increasing amounts of stretch. For repeated injury studies, cultures received a second injury 1 h after the initial insult. Repeated mild injury caused a slight increase in PrI uptake compared with single injury at 15 min and 24 h post-injury, which was evident primarily in glial cells. However, the neurites of neurones in cultures that received repeated insults showed signs of damage that were not evident after a single mild injury. The release of neurone-specific enolase (NSE) and S-100b protein, two common clinical markers of CNS damage, was also measured following the repeated injuries paradigm. When measured at 6 h post-injury, both NSE and S-100b were found to be elevated after repeated mild injuries when compared with the single injury group. These results suggest that cells of the hippocampus may be susceptible to cumulative damage following repeated mild traumatic insults. Both glial cells and neurones appear to exhibit increased signs of damage after repetitive injury. To our knowledge, this study represents the first report on the effects of repeated mechanical insults on specific cells of the brain using an in vitro model system. The biochemical pathways of cellular degradation following repeated mild injuries may differ considerably from those that are activated by a single mild insult. Therefore, we hope to use this model in order to investigate secondary pathways of cellular damage after repeated mild traumatic injury, and as a rapid and economical means of screening possibilities for treatment strategies, including pharmaceutical intervention.