A novel method for the rapid detection of post-translationally modified visinin-like protein 1 in rat models of brain injury.

BACKGROUND: Although elevated serum levels of visinin-like protein 1 (VILIP-1), a neuron-specific calcium sensor protein, are associated with ischaemic stroke, only a single study has evaluated VILIP-1 as a biomarker of traumatic brain injury (TBI). The current proof-of-concept study was designed to determine whether serum VILIP-1 levels increase post-injury in a well-characterized rat unilateral cortical contusion model. METHODS: Lateral flow devices (LFDs) rapidly (< 20 min) detected trace serum levels (pg/mL) of VILIP-1 in a small input sample volume (10 µL). Temporal profiles of serum levels at baseline and post-injury were measured in male Sprague Dawley rats subjected to very mild-, mild unilateral-cortical contusion, or naïve surgery and in male Sprague Dawley rats following a diffuse TBI or sham surgery. RESULTS: Mean serum levels were significantly elevated by 0.5 h post-injury and remained so throughout the temporal profile compared with baseline in very mild and mild unilateral contusions but not in naïve surgeries. Serum levels were also elevated in a small cohort of animals subjected to a diffuse TBI injury. CONCLUSIONS: Overall, the current study demonstrates that the novel LFD is a reliable and rapid point-of-care diagnostic for the detection and quantification of serum levels of UB-VILIP-1 in a clinically relevant time frame.

Natural Compounds as a Therapeutic Intervention following Traumatic Brain Injury: The Role of Phytochemicals.

There has been a tremendous focus on the discovery and development of neuroprotective agents that might have clinical relevance following traumatic brain injury (TBI). This type of brain injury is very complex and is divided into two major components. The first component, a primary injury, occurs at the time of impact and is the result of the mechanical insult itself. This primary injury is thought to be irreversible and resistant to most treatments. A second component or secondary brain injury, is defined as cellular damage that is not immediately obvious after trauma, but that develops after a delay of minutes, hours, or even days. This injury appears to be amenable to treatment. Because of the complexity of the secondary injury, any type of therapeutic intervention needs to be multi-faceted and have the ability to simultaneously modulate different cellular changes. Because of diverse pharmaceutical interactions, combinations of different drugs do not work well in concert and result in adverse physiological conditions. Research has begun to investigate the possibility of using natural compounds as a therapeutic intervention following TBI. These compounds normally have very low toxicity and have reduced interactions with other pharmaceuticals. In addition, many natural compounds have the potential to target numerous different components of the secondary injury. Here, we review 33 different plant-derived natural compounds, phytochemicals, which have been investigated in experimental animal models of TBI. Some of these phytochemicals appear to have potential as possible therapeutic interventions to offset key components of the secondary injury cascade. However, not all studies have used the same scientific rigor, and one should be cautious in the interpretation of studies using naturally occurring phytochemical in TBI research.

Cognitive assessment of pycnogenol therapy following traumatic brain injury.

We have previously shown that pycnogenol (PYC) increases antioxidants, decreases oxidative stress, suppresses neuroinflammation and enhances synaptic plasticity following traumatic brain injury (TBI). Here, we investigate the effects of PYC on cognitive function following a controlled cortical impact (CCI). Adult Sprague-Dawley rats received a CCI injury followed by an intraperitoneal injection of PYC (50 or 100mg/kg). Seven days post trauma, subjects were evaluated in a Morris water maze (MWM) and evaluated for changes in lesion volume. Some animals were evaluated at 48h for hippocampal Fluoro-jade B (FJB) staining. The highest dose of PYC therapy significantly reduced lesion volume, with no improvement in MWM compared to vehicle controls. PYC failed to reduce the total number of FJB positive neurons in the hippocampus. These results suggest that the reduction of oxidative stress and neuroinflammation are not the key components of the secondary injury that contribute to cognitive deficits following TBI.

Molecular and cellular pathophysiology of preclinical Alzheimer's disease.

Although the two pathological hallmarks of Alzheimer's disease (AD), senile plaques composed of amyloid-ß (Aß) peptides and neurofibrillary tangles (NFTs) consisting of hyperphosphorylated tau, have been studied extensively in postmortem AD and relevant animal and cellular models, the pathogenesis of AD remains unknown, particularly in the early stages of the disease where therapies presumably would be most effective. We and others have demonstrated that Aß plaques and NFTs are present in varying degrees before the onset and throughout the progression of dementia. In this regard, aged people with no cognitive impairment (NCI), mild cognitive impairment (MCI, a presumed prodromal AD transitional state, and AD all present at autopsy with varying levels of pathological hallmarks. Cognitive decline, a requisite for the clinical diagnosis of dementia associated with AD, generally correlates better with NFTs than Aß plaques. However, correlations are even higher between cognitive decline and synaptic loss. In this review, we illustrate relevant clinical pathological research in preclinical AD and throughout the progression of dementia in several areas including Aß and tau pathobiology, single population expression profiling of vulnerable hippocampal and basal forebrain neurons, neuroplasticity, neuroimaging, cerebrospinal fluid (CSF) biomarker studies and their correlation with antemortem cognitive endpoints. In each of these areas, we provide evidence for the importance of studying the pathological hallmarks of AD not in isolation, but rather in conjunction with other molecular, cellular, and imaging markers to provide a more systematic and comprehensive assessment of the multiple changes that occur during the transition from NCI to MCI to frank AD.

Oxidative stress and hippocampal synaptic protein levels in elderly cognitively intact individuals with Alzheimer's disease pathology.

Neuritic amyloid plaques and neurofibrillary tangles are hallmarks of Alzheimer's disease (AD) and are major components used for the clinical diagnosis of this disorder. However, many individuals with no cognitive impairment (NCI) also present at autopsy with high levels of these AD pathologic hallmarks. In this study, we evaluated 15 autopsy cases from NCI individuals with high levels of AD-like pathology (high pathology no cognitive impairment) and compared them to age- and postmortem-matched cohorts of individuals with amnestic mild cognitive impairment and NCI cases with low AD-like pathology (low pathology no cognitive impairment [LPNCI]). Individuals classified as high pathology no cognitive impairment or amnestic mild cognitive impairment had a significant loss of both presynaptic and postsynaptic proteins in the hippocampus compared with those in the LPNCI cohort. In addition, these 2 groups had a significant increase in 3 different markers of oxidative stress compared with that in the LPNCI group. The changes in levels of synaptic proteins are strongly associated with levels of oxidative stress. These data suggest that cognitively older subjects without dementia but with increased levels of AD-like pathology may represent a very early preclinical stage of AD.

Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury.

Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT: Similar to microglia, astrocytes become strongly "activated" with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin-dependent activation of the transcription factor NFAT (Nuclear Factor of Activated T cells) selectively, we have shown that activated astrocytes contribute to neural dysfunction in animal models characterized by progressive/chronic neuropathology. Here, we show that the suppression of astrocytic calcineurin/NFATs helps to protect synaptic function and plasticity in an animal model in which pathology arises from a single traumatic brain injury. The findings suggest that at least some astrocyte functions impair recovery after trauma and may provide druggable targets for treating victims of acute nervous system injury.

Pycnogenol protects CA3-CA1 synaptic function in a rat model of traumatic brain injury.

Pycnogenol (PYC) is a patented mix of bioflavonoids with potent anti-oxidant and anti-inflammatory properties. Previously, we showed that PYC administration to rats within hours after a controlled cortical impact (CCI) injury significantly protects against the loss of several synaptic proteins in the hippocampus. Here, we investigated the effects of PYC on CA3-CA1 synaptic function following CCI. Adult Sprague-Dawley rats received an ipsilateral CCI injury followed 15 min later by intravenous injection of saline vehicle or PYC (10 mg/kg). Hippocampal slices from the injured (ipsilateral) and uninjured (contralateral) hemispheres were prepared at seven and fourteen days post-CCI for electrophysiological analyses of CA3-CA1 synaptic function and induction of long-term depression (LTD). Basal synaptic strength was impaired in slices from the ipsilateral, relative to the contralateral, hemisphere at seven days post-CCI and susceptibility to LTD was enhanced in the ipsilateral hemisphere at both post-injury timepoints. No interhemispheric differences in basal synaptic strength or LTD induction were observed in rats treated with PYC. The results show that PYC preserves synaptic function after CCI and provides further rationale for investigating the use of PYC as a therapeutic in humans suffering from neurotrauma.

Synaptic change in the posterior cingulate gyrus in the progression of Alzheimer's disease.

Mild cognitive impairment (MCI) is considered to be an early stage in the progression of Alzheimer's disease (AD) providing an opportunity to investigate brain pathogenesis prior to the onset of dementia. Neuroimaging studies have identified the posterior cingulate gyrus (PostC) as a cortical region affected early in the onset of AD. This association cortex is involved in a variety of different cognitive tasks and is intimately connected with the hippocampal/entorhinal cortex region, a component of the medial temporal memory circuit that displays early AD pathology. We quantified the total number of synapses in lamina 3 of the PostC using unbiased stereology coupled with electron microscopy from short postmortem autopsy tissue harvested from cases at different stage of AD progression. Individuals in the early stages of AD showed a significant decline in synaptic numbers compared to individuals with no cognitive impairment (NCI). Subjects with MCI exhibited synaptic numbers that were between the AD and NCI cohorts. Adjacent tissue was evaluated for changes in both pre and postsynaptic proteins levels. Individuals with MCI demonstrated a significant loss in presynaptic markers synapsin-1 and synaptophysin and postsynaptic markers PSD-95 and SAP-97. Levels of [3H]PiB binding was significantly increased in MCI and AD and correlated strongly with levels of synaptic proteins. All synaptic markers showed a significant association with Mini-Mental Status Examination scores. These results support the idea that the PostC synaptic function is affected during the prodromal stage of the disease and may underlie some of the early clinical sequelae associated with AD.

Resilience of precuneus neurotrophic signaling pathways despite amyloid pathology in prodromal Alzheimer's disease.

BACKGROUND: Reduction of precuneus choline acetyltransferase activity co-occurs with greater beta-amyloid (Aß) in Alzheimer's disease (AD). Whether this cholinergic deficit is associated with alteration in nerve growth factor (NGF) signaling and its relation to Aß plaque and neurofibrillary tangle (NFT) pathology during disease onset is unknown. METHODS: Precuneus NGF upstream and downstream signaling levels relative to Aß and NFT pathology were evaluated using biochemistry and histochemistry in 62 subjects with a premortem diagnosis of non-cognitively impaired (NCI; n = 23), mild cognitive impairment (MCI; n = 21), and mild to moderate AD (n = 18). RESULTS: Immunoblots revealed increased levels of proNGF in AD subjects but not MCI subjects, whereas cognate receptors were unchanged. There were no significant differences in protein level for the downstream survival kinase-signaling proteins Erk and phospho-Erk among groups. Apoptotic phospho-JNK, phospho-JNK/JNK ratio, and Bcl-2 were significantly elevated in AD subjects. Soluble Aß1-42 and fibrillar Aß measured by [(3)H] Pittsburgh compound-B ([(3)H]PiB) binding were significantly higher in AD subjects compared with MCI and NCI subjects. The density of plaques showed a trend to increase, but only 6-CN-PiB-positive plaques reached significance in AD subjects. AT8-positive, TOC-1-positive, and Tau C3-positive NFT densities were unchanged, whereas only AT8-positive neuropil thread density was statistically higher in AD subjects. A negative correlation was found between proNGF, phospho-JNK, and Bcl-2 levels and phospho-JNK/JNK ratio and cognition, whereas proNGF correlated positively with 6-CN-PiB-positive plaques during disease progression. CONCLUSIONS: Data indicate that precuneus neurotrophin pathways are resilient to amyloid toxicity during the onset of AD.

Evidence for Alzheimer's disease-linked synapse loss and compensation in mouse and human hippocampal CA1 pyramidal neurons.

Alzheimer's disease (AD) is associated with alterations in the distribution, number, and size of inputs to hippocampal neurons. Some of these changes are thought to be neurodegenerative, whereas others are conceptualized as compensatory, plasticity-like responses, wherein the remaining inputs reactively innervate vulnerable dendritic regions. Here, we provide evidence that the axospinous synapses of human AD cases and mice harboring AD-linked genetic mutations (the 5XFAD line) exhibit both, in the form of synapse loss and compensatory changes in the synapses that remain. Using array tomography, quantitative conventional electron microscopy, immunogold electron microscopy for AMPARs, and whole-cell patch-clamp physiology, we find that hippocampal CA1 pyramidal neurons in transgenic mice are host to an age-related synapse loss in their distal dendrites, and that the remaining synapses express more AMPA-type glutamate receptors. Moreover, the number of axonal boutons that synapse with multiple spines is significantly reduced in the transgenic mice. Through serial section electron microscopic analyses of human hippocampal tissue, we further show that putative compensatory changes in synapse strength are also detectable in axospinous synapses of proximal and distal dendrites in human AD cases, and that their multiple synapse boutons may be more powerful than those in non-cognitively impaired human cases. Such findings are consistent with the notion that the pathophysiology of AD is a multivariate product of both neurodegenerative and neuroplastic processes, which may produce adaptive and/or maladaptive responses in hippocampal synaptic strength and plasticity.

A time course of NADPH-oxidase up-regulation and endothelial nitric oxide synthase activation in the hippocampus following neurotrauma.

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase; NOX) is a complex enzyme responsible for increased levels of reactive oxygen species (ROS), superoxide (O2(•-)). NOX-derived O2(•-) is a key player in oxidative stress and inflammation-mediated multiple secondary injury cascades (SIC) following traumatic brain injury (TBI). The O2(•-) reacts with nitric oxide (NO), produces various reactive nitrogen species (RNS), and contributes to apoptotic cell death. Following a unilateral cortical contusion, young adult rats were killed at various times postinjury (1, 3, 6, 12, 24, 48, 72, and 96 h). Fresh tissue from the hippocampus was analyzed for NOX activity, and level of O2(•-). In addition we evaluated the translocation of cytosolic NOX proteins (p67(Phox), p47(Phox), and p40(Phox)) to the membrane, along with total NO and the activation (phosphorylation) of endothelial nitric oxide synthase (p-eNOS). Results show that both enzymes and levels of O2(•-) and NO have time-dependent injury effects in the hippocampus. Translocation of cytosolic NOX proteins into membrane, NOX activity, and O2(•-) were also increased in a time-dependent fashion. Both NOX activity and O2(•-) were increased at 6 h. Levels of p-eNOS increased within 1h, with significant elevation of NO at 12h post-TBI. Levels of NO failed to show a significant association with p-eNOS, but did associate with O2(•-). NOX up-regulation strongly associated with both the levels of O2(•-) and the total NO. The initial 12 h post-TBI are very important as a possible window of opportunity to interrupt SIC. It may be important to selectively target the translocation of cytosolic subunits for the modulation of NOX function.

Development of a database for translational spinal cord injury research.

Efforts to understand spinal cord injury (SCI) and other complex neurotrauma disorders at the pre-clinical level have shown progress in recent years. However, successful translation of basic research into clinical practice has been slow, partly because of the large, heterogeneous data sets involved. In this sense, translational neurological research represents a "big data" problem. In an effort to expedite translation of pre-clinical knowledge into standards of patient care for SCI, we describe the development of a novel database for translational neurotrauma research known as Visualized Syndromic Information and Outcomes for Neurotrauma-SCI (VISION-SCI). We present demographics, descriptive statistics, and translational syndromic outcomes derived from our ongoing efforts to build a multi-center, multi-species pre-clinical database for SCI models. We leveraged archived surgical records, postoperative care logs, behavioral outcome measures, and histopathology from approximately 3000 mice, rats, and monkeys from pre-clinical SCI studies published between 1993 and 2013. The majority of animals in the database have measures collected for health monitoring, such as weight loss/gain, heart rate, blood pressure, postoperative monitoring of bladder function and drug/fluid administration, behavioral outcome measures of locomotion, and tissue sparing postmortem. Attempts to align these variables with currently accepted common data elements highlighted the need for more translational outcomes to be identified as clinical endpoints for therapeutic testing. Last, we use syndromic analysis to identify conserved biological mechanisms of recovery after cervical SCI between rats and monkeys that will allow for more-efficient testing of therapeutics that will need to be translated toward future clinical trials.

A distinct subfraction of Aß is responsible for the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain.

The positron emission tomography (PET) ligand (11) C-labeled Pittsburgh compound B (PIB) is used to image ß-amyloid (Aß) deposits in the brains of living subjects with the intent of detecting early stages of Alzheimer's disease (AD). However, deposits of human-sequence Aß in amyloid precursor protein transgenic mice and non-human primates bind very little PIB. The high stoichiometry of PIB:Aß binding in human AD suggests that the PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions. In this study, (3) H-PIB was employed to track purification of the PIB-binding site in > 90% yield from frontal cortical tissue of autopsy-diagnosed AD subjects. The purified PIB-binding site comprises a distinct, highly insoluble subfraction of the Aß in AD brain with low buoyant density because of the sodium dodecyl sulfate-resistant association with a limited subset of brain proteins and lipids with physical properties similar to lipid rafts and to a ganglioside:Aß complex in AD and Down syndrome brain. Both the protein and lipid components are required for PIB binding. Elucidation of human-specific biological components and pathways will be important in guiding improvement of the animal models for AD and in identifying new potential therapeutic avenues. A lipid-associated subpopulation of Aß accounts for the high-affinity binding of Pittsburgh compound B (PIB) in Alzheimer's disease brain. Mass spectrometry of the isolated PIB-binding site from frontal cortex identified Aß peptides and a set of plaque-associated proteins in AD but not age-matched normal brain. The PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions.

ABCC9 gene polymorphism is associated with hippocampal sclerosis of aging pathology.

Hippocampal sclerosis of aging (HS-Aging) is a high-morbidity brain disease in the elderly but risk factors are largely unknown. We report the first genome-wide association study (GWAS) with HS-Aging pathology as an endophenotype. In collaboration with the Alzheimer's Disease Genetics Consortium, data were analyzed from large autopsy cohorts: (#1) National Alzheimer's Coordinating Center (NACC); (#2) Rush University Religious Orders Study and Memory and Aging Project; (#3) Group Health Research Institute Adult Changes in Thought study; (#4) University of California at Irvine 90+ Study; and (#5) University of Kentucky Alzheimer's Disease Center. Altogether, 363 HS-Aging cases and 2,303 controls, all pathologically confirmed, provided statistical power to test for risk alleles with large effect size. A two-tier study design included GWAS from cohorts #1-3 (Stage I) to identify promising SNP candidates, followed by focused evaluation of particular SNPs in cohorts #4-5 (Stage II). Polymorphism in the ATP-binding cassette, sub-family C member 9 (ABCC9) gene, also known as sulfonylurea receptor 2, was associated with HS-Aging pathology. In the meta-analyzed Stage I GWAS, ABCC9 polymorphisms yielded the lowest p values, and factoring in the Stage II results, the meta-analyzed risk SNP (rs704178:G) attained genome-wide statistical significance (p = 1.4 × 10(-9)), with odds ratio (OR) of 2.13 (recessive mode of inheritance). For SNPs previously linked to hippocampal sclerosis, meta-analyses of Stage I results show OR = 1.16 for rs5848 (GRN) and OR = 1.22 rs1990622 (TMEM106B), with the risk alleles as previously described. Sulfonylureas, a widely prescribed drug class used to treat diabetes, also modify human ABCC9 protein function. A subsample of patients from the NACC database (n = 624) were identified who were older than age 85 at death with known drug history. Controlling for important confounders such as diabetes itself, exposure to a sulfonylurea drug was associated with risk for HS-Aging pathology (p = 0.03). Thus, we describe a novel and targetable dementia risk factor.

Is synaptic loss a unique hallmark of Alzheimer's disease?

Synapses may represent a key nidus for dementia including Alzheimer's disease (AD) pathogenesis. Here we review published studies and present new ideas related to the question of the specificity of synapse loss in AD. Currently, AD is defined by the regional presence of neuritic plaques and neurofibrillary tangles in the brain. The severity of involvement by those pathological hallmarks tends to correlate both with antemortem cognitive status, and also with synapse loss in multiple brain areas. Recent studies from large autopsy series have led to a new standard of excellence with regard to clinical-pathological correlation and to improved comprehension of the numerous brain diseases of the elderly. These studies have provided evidence that it is the rule rather than the exception for brains of aged individuals to demonstrate pathologies (often multiple) other than AD plaques and tangles. For many of these comorbid pathologies, the extent of synapse loss is imperfectly understood but could be substantial. These findings indicate that synapse loss is probably not a hallmark specific to AD but rather a change common to many diseases associated with dementia.

Hippocampal sclerosis of aging, a prevalent and high-morbidity brain disease.

Hippocampal sclerosis of aging (HS-Aging) is a causative factor in a large proportion of elderly dementia cases. The current definition of HS-Aging rests on pathologic criteria: neuronal loss and gliosis in the hippocampal formation that is out of proportion to AD-type pathology. HS-Aging is also strongly associated with TDP-43 pathology. HS-Aging pathology appears to be most prevalent in the oldest-old: autopsy series indicate that 5-30 % of nonagenarians have HS-Aging pathology. Among prior studies, differences in study design have contributed to the study-to-study variability in reported disease prevalence. The presence of HS-Aging pathology correlates with significant cognitive impairment which is often misdiagnosed as AD clinically. The antemortem diagnosis is further confounded by other diseases linked to hippocampal atrophy including frontotemporal lobar degeneration and cerebrovascular pathologies. Recent advances characterizing the neurocognitive profile of HS-Aging patients have begun to provide clues that may help identify living individuals with HS-Aging pathology. Structural brain imaging studies of research subjects followed to autopsy reveal hippocampal atrophy that is substantially greater in people with eventual HS-Aging pathology, compared to those with AD pathology alone. Data are presented from individuals who were followed with neurocognitive and neuroradiologic measurements, followed by neuropathologic evaluation at the University of Kentucky. Finally, we discuss factors that are hypothesized to cause or modify the disease. We conclude that the published literature on HS-Aging provides strong evidence of an important and under-appreciated brain disease of aging. Unfortunately, there is no therapy or preventive strategy currently available.

Dose- and time-dependent neuroprotective effects of Pycnogenol following traumatic brain injury.

After traumatic brain injury (TBI), both primary and secondary injury cascades are initiated, leading to neuronal death and cognitive dysfunction. We have previously shown that the combinational bioflavonoid, Pycnogenol (PYC), alters some secondary injury cascades and protects synaptic proteins when administered immediately following trauma. The purpose of the present study was to explore further the beneficial effects of PYC and to test whether it can be used in a more clinically relevant fashion. Young adult male Sprague-Dawley rats were subjected to a unilateral moderate/severe cortical contusion. Subjects received a single intravenous (i.v.) injection of PYC (1, 5, or 10 mg/kg) or vehicle, with treatment initiated at 15 min, 2 h, or 4 h post injury. All rats were killed at 96 h post TBI. Both the cortex and hippocampus ipsilateral and contralateral to the injury were evaluated for possible changes in oxidative stress (thiobarbituric acid reactive species; TBARS) and both pre- and post-synaptic proteins (synapsin-I, synaptophysin, drebrin, post synaptic density protein-95, and synapse associated protein-97). Following TBI, TBARS were significantly increased in both the injured cortex and ipsilateral hippocampus. Regardless of the dose and delay in treatment, PYC treatment significantly lowered TBARS. PYC treatment significantly protected both the cortex and hippocampus from injury-related declines in pre- and post-synaptic proteins. These results demonstrate that a single i.v. treatment of PYC is neuroprotective after TBI with a therapeutic window of at least 4 h post trauma. The natural bioflavonoid PYC may provide a possible therapeutic intervention in neurotrauma.

Synapse stability in the precuneus early in the progression of Alzheimer's disease.

Amnestic mild cognitive impairment (aMCI) is considered to be one of the early stages in the progression from no cognitive impairment (NCI) to Alzheimer's disease (AD). Individuals with aMCI have increased levels of AD-type neuropathology in multiple regions of the neocortex and hippocampus and demonstrate a loss of synaptic connectivity. Recent neuroimaging studies have reported increased levels of 11C-PiB (Pittsburgh, compound B) in regions of the neocortex including the precuneus region of the medial parietal lobe. This cortical region has been implicated in episodic memory, which is disrupted early in the progression of AD. In this study, unbiased stereology coupled with electron microscopy was used to quantify total synaptic numbers in lamina 3 of the precuneus from short postmortem autopsy tissue harvested from subjects who died at different cognitive stages during the progression of AD. Individuals with aMCI did not reveal a statistically significant decline in total synapses compared to the NCI cohort while the AD group did show a modest but significant decline. Synaptic numbers failed to correlate with several different cognitive tasks including the Mini-Mental State Examination scores and episodic memory scores. Although levels of [3H]PiB binding were elevated in both the aMCI and AD groups, it did not strongly correlate with synaptic counts. These results support the idea that despite increased amyloid load, the precuneus region does not show early changes in synaptic decline during the progression of AD.

Neuroprotective effect of Pycnogenol® following traumatic brain injury.

Traumatic brain injury (TBI) involves primary and secondary injury cascades that underlie delayed neuronal dysfunction and death. Oxidative stress is one of the most celebrated secondary injury mechanisms. A close relationship exists between levels of oxidative stress and the pathogenesis of TBI. However, other cascades, such as an increase in proinflammatory cytokines, also play important roles in the overall response to the trauma. Pharmacologic intervention, in order to be successful, requires a multifaceted approach. Naturally occurring flavonoids are unique in possessing not only tremendous free radical scavenging properties but also the ability to modulate cellular homeostasis leading to a reduction in inflammation and cell toxicity. This study evaluated the therapeutic role of Pycnogenol (PYC), a patented combinational bioflavonoid. Young adult Sprague-Dawley rats were subjected to a unilateral moderate cortical contusion and treated post injury with PYC or vehicle. At either 48 or 96 h post trauma, the animals were killed and the cortex and hippocampus analyzed for changes in enzymatic and non-enzymatic oxidative stress markers. In addition, possible changes in both pre- and post-synaptic proteins (synapsin-1, PSD-95, drebrin, synapse associated protein-97) were analyzed. Finally, a separate cohort of animals was used to evaluate two proinflammatory cytokines (IL-6, TNF-α). Following the trauma there was a significant increase in oxidative stress in both the injured cortex and the ipsilateral hippocampus. Animals treated with PYC significantly ameliorated levels of protein carbonyls, lipid peroxidation, and protein nitration. The PYC treatment also significantly reduced the loss of key pre- and post-synaptic proteins with some levels in the hippocampus of PYC treated animals not significantly different from sham operated controls. Although levels of the proinflammatory cytokines were significantly elevated in both injury groups, the cohort treated with PYC showed a significant reduction compared to vehicle treated controls. These results are the first to show a neuroprotective effect of PYC following TBI. They also suggest that the diverse effects of bioflavonoids may provide a unique avenue for possible therapeutic intervention following head trauma.

Hippocampal proNGF signaling pathways and ß-amyloid levels in mild cognitive impairment and Alzheimer disease.

Hippocampal precursor of nerve growth factor (proNGF)/NGF signaling occurs in conjunction with ß-amyloid (Aß) accumulations in Alzheimer disease (AD). To assess the involvement of this pathway in AD progression, we quantified these proteins and their downstream pathway activators in postmortem tissues from the brains of subjects with no cognitive impairment (NCI), mild cognitive impairment (MCI), and AD using immunoblotting and ELISA. Hippocampal proNGF was significantly greater in AD cases compared with those in NCI and MCI cases. TrkA was significantly reduced in MCI compared with those in NCI and AD, whereas p75 neurotrophin receptor, sortilin, and neurotrophin receptor homolog 2 remained stable. Akt decreased from NCI to MCI to AD, whereas phospho-Akt and phospho-Akt-to-Akt ratio were elevated in AD compared with those in MCI and NCI. No differences were found in phospho-Erk, Erk, or their ratio across groups. Although c-jun kinase (JNK) remained stable across groups, phospho-JNK and the phospho-JNK-to-JNK ratio increased significantly in AD compared with those in NCI and MCI. Expression levels of Aß(1-40), Aß(1-42), and Aß(40/42) ratio were stable. Statistical analysis revealed a strong positive correlation between proNGF and phospho-JNK, although only proNGF was negatively correlated with cognitive function and only TrkA was negatively associated with pathologic criteria. These findings suggest that alterations in the hippocampal NGF signaling pathway in MCI and AD favor proNGF-mediated proapoptotic pathways, and that this is independent of Aß accumulation during AD progression.