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.

Hypertension refers to elevated blood pressure, a precursor to heart attack, stroke, and renal disease. Several drugs, such as angiotensin-converting enzyme (ACE) inhibitors, treat hypertension, but diet modifications can have a positive impact on hypertension, alone or coupled with therapeutics. A diet low in Na + and rich in vegetables and fruits, especially those containing flavonoids, is especially good for blood pressure. Blueberries contain anthocyanins and other flavonoids. Recent research from our lab has shown that feeding blueberries to experimental animals lowers blood pressure by inhibiting ACE and, in doing so, reduces hypertension-induced renal damage.

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 (P < 0.05). 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 (P < 0.05). 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.

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 25μM, 50μM or 100μM oxyresveratrol at the time of injury. Trauma produced marked neuronal death when measured 24h post-injury, and oxyresveratrol significantly inhibited this death. Microscopic examination of glia suggested signs of toxicity in cultures treated with 100μM oxyresveratrol, as demonstrated by elevated S-100B protein release and a high proportion of cells with condensed nuclei. Cultures exposed to glutamate (100μM) for 24h exhibited ~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.

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 < 0.05). Feeding a 3% blueberry diet for 2 weeks lowered ACE activity in the SHRSP (p < 0.05) but not the normotensive rats. ACE activity in plasma of SHRSP was no longer elevated at weeks 4 and 6, but blueberry feeding inhibited ACE in SHRSP after 6 weeks. Blueberry diets had no effect on ACE activity in lung, testis, kidney, or aorta. Our results suggest that dietary blueberries may be effective in managing early stages of hypertension, partially due to an inhibition of soluble ACE activity.

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.

Obesity and dyslipidemia are major risk factors for cardiovascular disease. To test the health effects of antioxidant‐enriched diets, high‐fat diets (20% by weight) were fed to control (C57BL/6J) and apolipoprotein E‐deficient (ApoE‐/‐) mice, a strain that develops atherosclerotic lesions. Mice were fed either normal AIN‐93 purified diet (CON) or one that contained 5% freeze‐dried cranberry (Vaccinium macrocarpon; CB) for 8 weeks. There was less weight gain in animals fed CB from weeks 3 through 8 (p<0.05; two‐way ANOVA). At week 8, control mice fed CON weighed 39.4 ± 1.95 g, compared to 35.4 ± 1.55 g for control mice fed CB. Similarly, at week 8, ApoE‐/‐ mice fed CON weighed 40.5 ± 1.22 g, compared to 37.5 ± 1.60 g for those fed CB. The amount of abdominal white adipose tissue was lower in CB‐fed mice. Diets were found to be isocaloric, and there were no differences measured in the levels of carbohydrates, fats, cholesterol, and protein in the 2 diets. ApoE‐/‐ mice had elevated plasma cholesterol, HDL, and triglyceride values (p<0.0001), but diet had no effect on any of these endpoints. Plasma pyruvate, lactate, and keto acids will be measured to determine a potential metabolic mechanism for reduced weight gain. These data suggest that CB diet may improve energy balance which would benefit cardiovascular disease and obesity. (Funded by AIF and NSERC).

We have shown that feeding cranberries (Vaccinium. macrocarpon, CB) prolongs survival in spontaneously hypertensive stroke‐prone rats (SHRSP). Here, we evaluated the effect of feeding blueberries (V. angustifolium, BB) on survival and evaluated possible mechanisms. SHRSP were fed control diet or a diet enriched with 3% CB or 3% BB until the onset of stroke. Weight, feed intake, and systolic blood pressure (BP) were monitored weekly; urinary F2‐isoprostanes, a marker of oxidative stress, were measured at the end of the study. BB lowered BP by 6‐10% at weeks 5‐7, whereas CB lowered BP by 10% only at week 5. BB did not prolong survival, whereas CB increased survival by 40%. Rats with high BP at wks 4 and 5 were more likely to demonstrate early stroke symptoms (p<0.01). Feed efficiency over the first 5 wks also was positively correlated with days to stroke onset (p=0.018, control; p=0.052, CB). Weight at wk 5 was positively correlated with days to stroke in CB (p=0.038), but not in control‐fed SHRSP (p=0.41). In CB‐fed SHRSP, but not control rats, F2 isoprostanes were negatively correlated with BP at wk 5 (p=0.034), and positively correlated with days to stroke (p=0.027). These results suggest that effects on BP and weight/ feed efficiency can contribute to prolongation of stroke onset in SHRSP. Since F2‐isoprostanes are markers of lipid peroxidation, it is surprisingly that their levels promote survival. (Funded by AIF and NSERC).

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 overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a common underlying mechanism of many neuropathologies, as they have been shown to damage various cellular components, including proteins, lipids and DNA. Free radicals, especially superoxide (O(2)*-), and non-radicals, such as hydrogen peroxide (H(2)O(2)), can be generated in quantities large enough to overwhelm endogenous protective enzyme systems, such as superoxide dismutase (SOD) and reduced glutathione (GSH). Here we review the mechanisms of ROS and RNS production, and their roles in ischemia, traumatic brain injury and aging. In particular, we discuss several acute and chronic pharmacological therapies that have been extensively studied in order to reduce ROS/RNS loads in cells and the subsequent oxidative stress, so-called "free-radical scavengers." Although the overall aim has been to counteract the detrimental effects of ROS/RNS in these pathologies, success has been limited, especially in human clinical studies. This review highlights some of the recent successes and failures in animal and human studies by attempting to link a compound's chemical structure with its efficacy as a free radical scavenger. In particular, we demonstrate how antioxidants derived from natural products, as well as long-term dietary alterations, may prove to be effective scavengers of ROS and RNS.

Selecting appropriate research models is of utmost importance as incorrect models can lead to misleading results. The spontaneously hypertensive stroke‐prone rat (SHRSP) is a commonly used model of cerebrovascular disease and hypertension. Signs of stroke are reported to be observed in SHRSP typically by age 14 weeks, involving any or all of the following acute symptoms: loss of coordination, twitching, weight loss, loss of appetite, and lack of thriftiness. However, after feeding a standard AIN93G purified diet, and 1 or 2% NaCl in water for 8 or 17 weeks, none of the symptoms of stroke were observed in any SHRSP in our laboratory (n=15). Thus, a follow‐up pilot study compared the incidence of stroke in SHRSP fed AIN93‐G purified diet to a commercially available rat chow starting at age 8 weeks. All rats had 4% NaCl in the diet. All SHRSP fed chow showed stroke symptoms at age 14.6 ± 0.7 wks (n=3), while no symptoms developed in SHRSP fed purified diet, and so rats were sacrificed at age 19 weeks (n=3). Body weight was significantly lower at age 14 wks in SHRSP fed chow (261.6 ± 7.5 g) than SHRSP fed AIN93‐G (307.3 ± 4.9 g). In another study, SHRSP rats fed chow with 4 % NaCl developed stroke symptoms by age 15.3 ± 0.5 wks (n=10). These data provide evidence to suggest that dietary composition is a major contributor to the onset of stroke in SHRSP. Further investigations are being carried‐out to determine the specific dietary components of the commercial rat chow that increase incidence of stroke in SHRSP. Funded by NSERC & Atlantic Innovation Fund.

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 minor traumatic brain injury (mTBI) in sports is often looked at as a bagatelle. The treating physician underestimates the severity of the injury suspecting that a mTBI is a nonstructural lesion with an overall excellent prognosis in the majority of the cases.This paper shows that the minor traumatic brain injury may be a structural brain lesion with potentially life-threatening dangers. The therapy should follow exactly defined guidelines, e.g., stepwise protocol of the Concussion in Sports (CIS-) Group. Return to sports activities should happen only when all physical but also cognitive symptoms have subsided. All mTBIs that have been sustained prior to the actual injury have to be recorded properly because repeated mTBIs may cause chronic degenerative brain damage.Neuropsychological testing will aid in the correct diagnosis of a mTBI and is a useful parameter in the course of the injury. In the future biochemical markers may serve as indicators of the severity of the brain injury and may also aid in predicting the outcome after TBI. Today biochemical markers do not serve as a substitute for neuroimaging.

La survenue d’un nouveau traumatisme craniocérébral au décours d’un traumatisme craniocérébral léger (minor traumatic brain injury, mTBI) entraîne non seulement une péjoration des symptômes, mais peut aussi conduire, dans des cas pas si rares que cela, à une chronification de ces derniers. Il est par conséquent extrêmement important de rechercher la présence, dans l’anamnèse, d’antécédents éventuels de traumatismes craniocérébraux légers. Les tests neuropsychologiques contribuent au diagnostic de mTBI et constituent des paramètres précieux pour suivre l’évolution de la situation. Certains marqueurs biochimiques pourraient être utilisés, dans un avenir proche, tant dans une optique diagnostique que pronostique. Pour l’instant, ils ne sauraient cependant prétendre à remplacer les examens neuroradiologiques. Un mTBI survenant chez l’enfant nécessite le recours à un algorithme de prise en charge particulier, dans la mesure où chez celui-ci, des forces plus importantes entraînent des symptômes moins prononcés que chez l’adulte. De plus, les symptômes pathologiques chez l’enfant sont souvent difficiles à distinguer avec certitude d’une symptomatologie non pathologique. On ne recommandera jamais assez le port des éléments de protection obligatoires dans certains sports pour prévenir les traumatismes craniocérébraux. La recherche d’améliorations dans ce domaine mérite d’être encouragée. Le recours à des mentonnières protectrices pour la mâchoire peut prévenir les mTBI dans de nombreux cas de traumatismes frontaux.

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.

Schlüsselwörter Gehirnerschütterung - leichtes Schädel-Hirn-Trauma - Sportverletzung - S-100 Protein - Stufenschema

Recent evidence suggests repeated mild brain trauma may result in cumulative damage. We investigated cell damage and death in hippocampal cultures following repeated mechanical trauma in vitro by measuring propidium iodide (PrI) uptake, release of neuron-specific enolase (NSE) and glial S-100β protein, and performing neuronal counts. Cultures receiving two mild injuries (31% stretch) 1 or 24 h apart displayed different profiles of PrI uptake and S-100β release, although neuronal loss and NSE release was similar in both paradigms. Cells receiving a subthreshold, low-level stretch (10%) repeated several times eventually stained with PrI. Cultures administered 10% stretch before mild injury released less S-100β than mild injury alone, suggesting a preconditioning effect. Lastly, exogenous S-100β applied to injured cultures decreased PrI uptake, implying a protective role. These results suggest cumulative damage is dependent on injury severity and inter-injury interval, and that neurons and glia react differently to various injury paradigms.