Autophagy and protein aggregation as a mechanism of dopaminergic degeneration in a primary human dopaminergic neuronal model.

The pathophysiology underlying the loss of dopaminergic neurons in Parkinson's disease (PD) is unclear. A gap of knowledge in the molecular and cellular events leading to degeneration of the nigrostriatal DA system is a major barrier to the development of effective therapies for PD. 1-methyl-4-phenylpyridinium (MPP+) is used as a reliable in vitro model of PD in dopaminergic neurons; however, the molecular mechanisms that lead to cell death with this model are not fully understood. Additionally, there is a lack of translational in vitro models to fully understand progressive dopaminergic neurotoxicity. Here, we propose cultures of primary human dopaminergic neuronal precursor cells (HDNPCs) as a model to study progressive dopaminergic toxicity and neuronal damage in PD. We evaluated the concentration-response of MPP+ (0-10 mM) at 24 h, using cell viability and mitochondrial activity assays (LDH, XTT, Live/Dead staining, and MitoTracker). Based on concentration-response data, we chose two concentrations (1.0 and 2.5 mM) of MPP+ to evaluate markers of autophagy and dopaminergic status [tyrosine hydroxylase (TH)] after a 24-h exposure. Exposure to MPP+ induced cytotoxicity, reduced cell viability, and decreased mitochondrial activity. MPP+ at 1.0 and 2.5 mM also induced expression of lysosome-associated membrane protein 1 (LAMP-1) and increased the ratio of light chain 3 (LC3), LC3BII/LC3BI. The expression of TH also decreased. Furthermore, α-synuclein (α-SYN) and parkin were evaluated by immunofluorescence (IF) at 1.0 and 2.5 mM MPP+ after 24 h. A qualitative analysis revealed decreased parkin expression while α-SYN aggregation was observed in the cytoplasm and the nucleus. These data suggest that in HDNPCs MPP+ can cause cytotoxicity and neuronal damage. This damage may be mediated by autophagy, dopamine synthesis, and protein aggregation. The combination of HDNPCs and MPP+ may serve as valuable in vitro model of progressive dopaminergic neurotoxicity for research into potential treatments for PD.

Functional Analysis of the Cortical Transcriptome and Proteome Reveal Neurogenesis, Inflammation, and Cell Death after Repeated Traumatic Brain Injury In vivo.

The pathological effects of repeated traumatic brain injuries (TBIs) are largely unknown. To gain a detailed understanding of the cortical tissue acute biological response after one or two TBIs, we utilized RNA-sequencing and protein mass spectrometry techniques. Using our previously validated C57Bl/6 weight-drop model, we administered one or two TBIs of a mild or moderate severity. Double injury conditions were spaced 7 days apart, and cortical tissue was isolated 24 h after final injury. Analysis was carried out through functional gene annotation, utilizing Gene Ontology, for both the proteome and transcriptome. Major themes across the four different conditions include: neurogenesis; inflammation and immune response; cell death; angiogenesis; protein modification; and cell communication. Proteins associated with neurogenesis were found to be upregulated after single injuries. Transcripts associated with angiogenesis were upregulated in the moderate single, mild double, and moderate double TBI conditions. Genes associated with inflammation and immune response were upregulated in every condition, with the moderate single condition reporting the most functional groups. Proteins or genes involved in cell death, or apoptosis, were upregulated in every condition. Our results emphasize the significant differences found in proteomic and transcriptomic changes in single versus double injuries. Further, cortical omics analysis offers important insights for future studies aiming to deepen current knowledge on the development of secondary injuries and neurobehavioral impairments after brain trauma.

The Role of the Cerebellum in Repetitive Behavior Across Species: Childhood Stereotypies and Deer Mice.

Recent studies suggest that the cerebellum may have a significant role in repetitive behaviors. In primary complex motor stereotypies, typically developing children have repetitive movements usually involving rhythmic flapping/waving arm/hand movements. Similarly, the deer mouse animal model exhibits inherited repetitive behaviors, with increased frequencies of spontaneous jumping and rearing. In this study, data from both children with motor stereotypies and deer mice were used to investigate the role of the cerebellum in repetitive behaviors. The 3.0-T MRI volumetric imaging of the cerebellum was obtained in 20 children with primary complex motor stereotypies and 20 healthy controls. In deer mice, cerebellar volume (n = 7/group) and cell counts (n = 9/group) were compared between high- and low-activity animals. Levels of cerebellar neurotransmitters were also determined via HPLC (n = 10/group). In children with stereotypies, (a) there were a statistically significant reduction (compared to controls) in the white matter volume of the posterior cerebellar lobule VI-VII that negatively correlated with motor control and (b) an 8% increase in the anterior vermis gray matter that positively correlated with motor Stereotypy Severity Scores (SSS). In deer mice, (a) there was a significant increase in the volume of the anterior vermal granular cell layer that was associated with higher activity and (b) dentate nucleus cell counts were higher in high activity animals. Similar increases in volume were observed in anterior vermis in children with stereotypies and a deer mouse model of repetitive behaviors. These preliminary findings support the need for further investigation of the cerebellum in repetitive behaviors.

Acute MDPV Binge Paradigm on Mice Emotional Behavior and Glial Signature.

3,4-Methylenedioxypyrovalerone (MDPV), a widely available synthetic cathinone, is a popular substitute for classical controlled drugs of abuse, such as methamphetamine (METH). Although MDPV poses public health risks, its neuropharmacological profile remains poorly explored. This study aimed to provide evidence on that direction. Accordingly, C57BL/6J mice were exposed to a binge MDPV or METH regimen (four intraperitoneal injections every 2 h, 10 mg/kg). Locomotor, exploratory, and emotional behavior, in addition to striatal neurotoxicity and glial signature, were assessed within 18-24 h, a known time-window encompassing classical amphetamine dopaminergic neurotoxicity. MDPV resulted in unchanged locomotor activity (open field test) and emotional behavior (elevated plus maze, splash test, tail suspension test). Additionally, striatal TH (METH neurotoxicity hallmark), Iba-1 (microglia), GFAP (astrocyte), RAGE, and TLR2/4/7 (immune modulators) protein densities remained unchanged after MDPV-exposure. Expectedly, and in sheer contrast with MDPV, METH resulted in decrease general locomotor activity paralleled by a significant striatal TH depletion, astrogliosis, and microglia arborization alterations (Sholl analysis). This comparative study newly highlights that binge MDPV-exposure comes without evident behavioral, neurochemical, and glial changes at a time-point where METH-induced striatal neurotoxicity is clearly evident. Nevertheless, neuropharmacological MDPV signature needs further profiling at different time-points, regimens, and brain regions.

Effects of acetyl L-carnitine on zebrafish embryos: Phenotypic and gene expression studies.

Acetyl L-carnitine (ALCAR), a dietary supplement and an antioxidant, plays a vital role in the bioenergetic process that produces ATP. Although there are reports on antioxidant toxicity, there is no information on the potential toxicity of ALCAR. Here, using zebrafish embryos, we explored whether ALCAR modulated ATP synthesis, generation of reactive oxygen species (ROS) and expression of specific genes related to major signaling pathways that control metabolism, growth, differentiation, apoptosis and oxidative stress. First, we show that ALCAR elicits a physiologic response, as ATP levels increased after ALCAR treatment. Simultaneously, an increase in the expression of ROS, a by-product of ATP synthesis, was observed in the ALCAR-treated embryos. Consistent with higher ROS expression, the level of cysteine, a precursor of glutathione, was significantly reduced. ALCAR did not have any drastic effect on overall development and heart rate. Polymerase chain reaction-based gene expression array analyses showed no significant change in the expression of 83 genes related to 10 major signaling pathways including: the transforming growth factor ß (TGFß), Wingless and Int-1 (Wnt), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), Janus kinase/signal transducers and activators of transcription (JAK/STAT), p53, Notch, Hedgehog, Peroxisome proliferator-activated receptor (PPAR), oxidative stress, and hypoxia pathways. Our results show that the expression of 83 genes related to these major signaling pathways did not change significantly.

Nifedipine toxicity is exacerbated by acetyl l-carnitine but alleviated by low-dose ketamine in zebrafish in vivo.

Calcium channel blocker (CCB) poisoning is a common and sometimes life-threatening emergency. Our previous studies have shown that acetyl l-carnitine (ALCAR) prevents cardiotoxicity and developmental toxicity induced by verapamil, a CCB used to treat patients with hypertension. Here, we tested whether toxicities of nifedipine, a dihydropyridine CCB used to treat hypertension, can also be mitigated by co-treatment with ALCAR. In the zebrafish embryos at three different developmental stages, nifedipine induced developmental toxicity with pericardial sac edema in a dose-dependent manner, which were surprisingly exacerbated with ALCAR co-treatment. Even with low-dose nifedipine (5 µm), when the pericardial sac looked normal, ALCAR co-treatment showed pericardial sac edema. We hypothesized that toxicity by nifedipine, a vasodilator, may be prevented by ketamine, a known vasoconstrictor. Nifedipine toxicity in the embryos was effectively prevented by co-treatment with low (subanesthetic) doses (25-100 µm added to the water) of ketamine, although a high dose of ketamine (2 mm added to the water) partially prevented the toxicity.As expected of a CCB, nifedipine either in the presence or absence of ketamine-reduced metabolic reactive oxygen species (ROS), a downstream product of calcium signaling, in the rapidly developing digestive system. However, nifedipine induced ROS in the trunk region that showed significantly stunted growth indicating that the tissues under stress potentially produced pathologic ROS. To the best of our knowledge, these studies for the first time show that nifedipine and the dietary supplement ALCAR together induce adverse effects while providing evidence on the therapeutic efficacy of subanesthetic doses of ketamine against nifedipine toxicity in vivo.

Stretch-Induced Deformation as a Model to Study Dopaminergic Dysfunction in Traumatic Brain Injury.

Traumatic brain injury (TBI) is defined as damage to the brain that consequently disrupts normal function. Neuronal death, a hallmark of TBI, has been related to the development of neurodegenerative disorders like Parkinson's disease (PD), where loss of dopaminergic neurons and dopaminergic dysfunction are observed. To date, no in vitro model exists in which the dopaminergic damage observed in TBI is replicated. In this study, we evaluated the effects of in vitro simulated TBI on human dopaminergic neurons. To simulate TBI, neurons were subjected to 0%, 5%, 10%, 15%, 25% and 50% deformation. 24 h after injury, cell viability and apoptosis were determined by lactate dehydrogenase (LDH) release and DNA fragmentation, as well as ethidium homodimer and caspase 3/7 staining. Dopamine (DA) levels were determined by ELISA. Levels of tyrosine hydroxylase (TH) and DA transporter (DAT) were determined by western blot. Only 50% stretch increased LDH release and ethidium homodimer staining, suggesting the induction of necrosis. On the contrary, 25% and 50% stretch increased DNA fragmentation while 15%, 25% and 50% increased caspase 3/7 staining, suggesting that moderate and severe TBI promote apoptosis. Levels of intracellular DA decreased in a stretch-dependent manner with 15%, 25% and 50% stretch, which were related with a decrease in TH expression. Extracellular DA levels increased only at 50%. Levels of DAT remained unchanged regardless of treatment. These data support the use of stretch as a model to simulate TBI in vitro in human dopaminergic neurons, replicating the acute effects of TBI in the dopaminergic system.

Amyloid Beta 25-35 induces blood-brain barrier disruption in vitro.

The amyloid ß-peptide (Aß) is transported across the blood-brain barrier (BBB) by binding with the receptor for advanced glycation end products (RAGE). Previously, we demonstrated that the Aß fraction 25-35 (Aß25-35) increases RAGE expression in the rat hippocampus, likely contributing to its neurotoxic effects. However, it is still debated if the interaction of Aß with RAGE compromises the BBB function in Alzheimer' disease (AD). Here, we evaluated the effects of Aß25-35 in an established in vitro model of the BBB. Rat brain microvascular endothelial cells (rBMVECs) were treated with 20 µM active Aß25-35 or the inactive Aß35-25 (control), for 24 h. Exposure to Aß25-35 significantly decreased cell viability, increased cellular necrosis, and increased the production of reactive oxygen species (ROS), which triggered a decrease in the enzyme glutathione peroxidase when compared to the control condition. Aß25-35 also increased BBB permeability by altering the expression of tight junction proteins (decreasing zonula occludens-1 and increasing occludin). Aß25-35 induced monolayer disruption and cellular disarrangement of the BBB, with RAGE being highly expressed in the zones of disarrangement. Together, these data suggest that Aß25-35-induces toxicity by compromising the functionality and integrity of the BBB in vitro. Graphical abstract Aß25-35 induces BBB dysfunction in vitro, wich is likely mediated by OS and ultimately leads to disruption of BBB integrity and cell death.

An Alternative In Vitro Method for Examining Nanoparticle-Induced Cytotoxicity.

Conventional in vitro assays are often used as initial screens to identify potential toxic effects of nanoparticles (NPs). However, many NPs have shown interference with conventional in vitro assays, resulting in either false-positive or -negative outcomes. Here, we report an alternative method for the in vitro assessment of NP-induced cytotoxicity utilizing Fluoro-Jade C (FJ-C). To provide proof of concept and initial validation data, Ag-NPs and Au-NPs were tested in 3 different cell cultures including rat brain microvessel endothelial cells, mouse neural stem cells, and the human SH-SY5Y cell line. Conventional 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and lactate dehydrogenase (LDH) assays were run in parallel with the new method and served as references. The results demonstrate for the first time that FJ-C labeling can be a useful tool for assessing NP-induced cytotoxicity in vitro. Using these approaches, it was also demonstrated that removal of Ag-NPs-while keeping the Ag-ions that were released from the Ag-NPs in culture media-abolished the measured cytotoxicity, indicating that Ag-NPs rather than Ag-ions in solution contributed to the observed cytotoxic effects. Further, co-treatment of Ag-NPs with N-acetyl cysteine (NAC) prevented the observed cytotoxicity, suggesting a protective role of NAC in Ag-NP-induced cytotoxicity. Thus, this alternative in vitro assay is well suited for identify potential cytotoxicity associated with exposure to NPs.

Ketamine-induced attenuation of reactive oxygen species in zebrafish is prevented by acetyl l-carnitine in vivo.

Ketamine, an anesthetic, is a non-competitive antagonist of the calcium-permeable N-methyl-d-aspartate (NMDA) receptor. High concentrations of ketamine have been implicated in cardiotoxicity and neurotoxicity. Often, these toxicities are thought to be mediated by reactive oxygen species (ROS). However, findings to the contrary showing ketamine reducing ROS in mammalian cells and neurons in vitro, are emerging. Here, we determined the effects of ketamine on ROS levels in zebrafish larvae in vivo. Based on our earlier studies demonstrating reduction in ATP levels by ketamine, we hypothesized that as a calcium antagonist, ketamine would also prevent ROS generation, which is a by-product of ATP synthesis. To confirm that the detected ROS in a whole organism, such as the zebrafish larva, is specific, we used diphenyleneiodonium (DPI) that blocks ROS production by inhibiting the NADPH Oxidases (NOX). Upon 20 h exposure, DPI (5 and 10 µM) and ketamine at (1 and 2 mM) reduced ROS in the zebrafish larvae in vivo. Using acetyl l-carnitine (ALCAR), a dietary supplement, that induces mitochondrial ATP synthesis, we show elevated ROS generation with increasing ALCAR concentrations. Combined, ketamine and ALCAR counter-balanced ROS generation in the larvae suggesting that ketamine and ALCAR have opposing effects on mitochondrial metabolism, which may be key to maintaining ROS homeostasis in the larvae and affords ALCAR the ability to prevent ketamine toxicity. These results for the first time show ketamine's antioxidative and ALCAR's prooxidative effects in a live vertebrate.

Characterization of Serum Exosomes from a Transgenic Mouse Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's Disease (AD) is the most common type of dementia characterized by amyloid plaques containing Amyloid Beta (Aß) peptides and neurofibrillary tangles containing tau protein. In addition to neuronal loss, Cerebral Amyloid Angiopathy (CAA) commonly occurs in AD. CAA is characterized by Aß deposition in brain microvessels. Recent studies have suggested that exosomes (cell-derived vesicles containing a diverse cargo) may be involved in the pathogenesis of AD. OBJECTIVE: Isolate and characterize brain-derived exosomes from a transgenic mouse model of AD that presents CAA. METHODS: Exosomes were isolated from serum obtained from 13-month-old wild type and AD transgenic female mice using an exosome precipitation solution. Characterization of exosomal proteins was performed by western blots and dot blots. RESULTS: Serum exosomes were increased in transgenic mice compared to wild types as determined by increased levels of the exosome markers flotillin and alix. High levels of neuronal markers were found in exosomes, without any difference any between the 2 groups. Markers for endothelial-derived exosomes were decreased in the transgenic model, while astrocytic-derived exosomes were increased. Exosome characterization showed increased levels of oligomeric Aß and oligomeric and monomeric forms tau on the transgenic animals. Levels of amyloid precursor protein were also increased. In addition, pathological and phosphorylated forms of tau were detected, but no difference was observed between the groups. CONCLUSION: These data suggest that monomeric and oligomeric forms of Aß and tau are secreted into serum via brain exosomes, most likely derived from astrocytes in the transgenic mouse model of AD with CAA. Studies on the implication of this event in the propagation of AD are underway.

Cytotoxicity profile of pristine graphene on brain microvascular endothelial cells.

Graphene-based nanomaterials hold the potential to be used in a wide variety of applications, including biomedical devices. Pristine graphene (PG) is an un-functionalized, defect-free type of graphene that could be used as a material for neural interfacing. However, the neurotoxic effects of PG, particularly to the blood-brain barrier (BBB), have not been fully studied. The BBB separates the brain tissue from the circulating substances in the blood and is essential to maintain the brain homeostasis. The principal components of the BBB are brain microvascular endothelial cells (BMVECs), which maintain a protectively low permeability due to the expression of tight junction proteins. Here we analyzed the effects of PG on BMVECs in an in vitro model of the BBB. BMVECs were treated with PG at 0, 10, 50 and 100 µg/mL for 24 hours and viability and functional analyses of BBB integrity were performed. PG increased lactate dehydrogenase release at 50 and 100 µg/mL, suggesting the induction of necrosis. Surprisingly, 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium (XTT) conversion was increased at 10 and 50 µg/mL. In contrast, XTT conversion was decreased at 100 µg/mL, suggesting the induction of cell death. In addition, 100 µg/mL PG increased DNA fragmentation, suggesting induction of apoptosis. At the same time, 50 and 100 µg/mL of PG increased the endothelial permeability, which corresponded with a decrease in the expression of the tight junction protein occludin at 100 µg/mL. In conclusion, these results suggest that PG negatively affects the viability and function of the BBB endothelial cells in vitro.

Utilization of Hospice Care in Patients With Acute Ischemic Stroke.

BACKGROUND:: A significant percentage of terminally ill patients are discharged to hospice care following a devastating stroke. OBJECTIVE:: We sought to determine the factors associated with hospital discharge to hospice care in a large cohort of patients with stroke. METHODS:: Using the institutional Get With The Guidelines-Stroke database, all consecutive patients with acute ischemic stroke (AIS) who were alive at discharge, from January 2009 until July 2015, were analyzed. Univariate and multivariable statistical analyses were performed to determine the factors associated with discharge to hospice care. RESULTS:: Of 2446 patients with AIS, 3.4% died and were excluded of remaining 2363 patients, and 4.2% were discharged to hospice care. Univariate analysis identified patients who were discharged to hospice care to be older, caucasian, Medicare or private insurance, have atrial fibrillation, heart failure and less often had diabetes mellitus or smoked. Altered mentation at presentation and urinary tract infection were more common in patients discharged to hospice. On multivariable analysis, patients transferred to hospice care were older (odds ratio [OR]: 1.04, 95% confidence interval [CI]: 1.01-1.07; P < .001), had a high National Institute of Health Stroke Scale (NIHSS; OR: 1.15, 95% CI: 1.10-1.20; P < .001), and altered mental status at presentation (OR: 2.42, 95% CI: 1.29-4.55; P < .001). CONCLUSION:: In our study, elderly patients with high NIHSS and altered mental status were identified as factors associated with transition to hospice care following AIS. Prospective studies on the optimal timing of initiation of these consults are needed.

Evaluation of a score for the prehospital distinction between cerebrovascular disease and stroke mimic patients.

BACKGROUND: Patients with a sudden onset of focal neurological deficits consistent with stroke, who turn out to have alternative conditions, have been labeled stroke mimics. AIMS: We assessed a recently validated telemedicine-based stroke mimic score (TeleStroke mimic score; TM-score) and individual patient characteristics with regard to its discriminative value between cerebrovascular disease and stroke mimic patients in the in-person, pre-hospital setting. METHODS: We evaluated patients cared for in a mobile stroke unit in Berlin, Germany. We investigated whether the TM-score (comprising six parameters), Face Arm Speech Time test, and individual patient characteristics were able to differentiate cerebrovascular disease from stroke mimic patients. RESULTS: We included 423 patients (299 (70.7%) cerebrovascular disease and 124 (29.3%) stroke mimic) in the final analysis. A TM-score > 30 indicated a high probability of a cerebrovascular disease and a score ≤15 of a stroke mimic. The TM-score performed well to identify stroke mimics (area under the curve of 0.74 under receiver-operating characteristic curve analysis). The cerebrovascular disease patients were older (74.8 vs. 69.8 years, p = 0.001), had more often severe strokes (NIHSS > 14 25.8% vs. 11.3%, p = 0.001), presented more often with weakness of the face (70.9% vs. 42.7%, p = 0.001) or arm (60.9% vs. 33.9%, p = 0.001), dysarthria (59.5% vs. 40.3%, p < 0.001), history of atrial fibrillation (38.1% vs. 21.0%, p = 0.001), arterial hypertension (78.9% vs. 53.2%, p < 0.001), and less often with seizure (0.7% vs. 21.0%, p < 0.001). CONCLUSIONS: The TM-score and certain patient characteristics can help paramedics and emergency physicians in the field to identify stroke mimic patients and select the most appropriate hospital destination.

Rates, Characteristics, and Outcomes of Patients Transferred to Specialized Stroke Centers for Advanced Care.

Background While many patients are transferred to specialized stroke centers for advanced acute ischemic stroke (AIS) care, few studies have characterized these patients. We sought to determine variation in the rates and differences in the baseline characteristics and clinical outcomes between AIS cases presenting directly to stroke centers' front door versus Transfer-Ins from another hospital. Methods and Results We analyzed 970 390 AIS cases in the Get With The Guidelines-Stroke registry from January 2010 to March 2014 to compare hospitals with high Transfer-In rates (≥15%) versus those with low Transfer-In rates (<5%) and to compare the front-door versus Transfer-In patients admitted to those hospitals with high Transfer-In rates (high Transfer-In hospitals). Of 970 390 patients discharged from 1646 hospitals, 87% initially presented via the emergency department versus 13% were a Transfer-In from another hospital. High Transfer-In hospitals had a median 31% Transfer-In rate among all stroke discharges, were larger, had higher annual AIS volume and intravenous tPA (tissue-type plasminogen activator) rates, and were more often Midwest teaching hospitals and stroke centers. Compared with front-door, Transfer-In patients were younger, more often white, had higher median National Institutes of Health Stroke Scale scores, less often hypertension and previous stroke/transient ischemic attack, and higher in-hospital mortality (7.9% versus 4.9%; standardized difference, 12.4%). After multivariable adjustment, Transfer-In patients had higher in-hospital mortality and discharge modified Rankin scale. Conclusions There is significant regional variability in the transfer of patients with AIS. Because Transfer-In patients seem to have worse short-term outcomes, these patients have the potential to negatively influence institutional mortality rates and should be accounted for explicitly in hospital risk-profiling measures.

Characterization of uniaxial high-speed stretch as an in vitro model of mild traumatic brain injury on the blood-brain barrier.

Traumatic brain injury (TBI) occurs when external mechanical forces induce brain damage as result of impact, penetration or rapid acceleration/deceleration that causes deformation of brain tissue. Depending on its severity, TBI can be classified as mild, moderate or severe and can lead to blood-brain barrier (BBB) dysfunction. In the present study, we evaluated the effects of uniaxial high-speed stretch (HSS) at 0, 5, 10 and 15% on a pure culture of primary rat brain endothelial cells as an in vitro model of TBI to the BBB. LDH release, viability and apoptosis analysis, expression of tight junction proteins and endothelial permeability were evaluated 24 h after a single stretch episode. HSS slightly increased cell death and apoptosis at 10 and 15%, while LDH release was increased only at 15% stretch. Occludin expression was increased at 10% stretch, while claudin-5 expression was increased at 5% stretch, which also decreased the endothelial permeability. In summary, 15% HSS induced low levels of cell death, consistent with mild TBI and very low percentages of HSS (5%) enhanced the BBB properties, promoting the formation of a stronger barrier. These data support the use of 15% HSS as valuable tool in the study of mild TBI to the BBB in vitro.

Validating the TeleStroke Mimic Score: A Prediction Rule for Identifying Stroke Mimics Evaluated Over Telestroke Networks.

BACKGROUND AND PURPOSE: Up to 30% of acute stroke evaluations are deemed stroke mimics, and these are common in telestroke as well. We recently published a risk prediction score for use during telestroke encounters to differentiate stroke mimics from ischemic cerebrovascular disease derived and validated in the Partners TeleStroke Network. Using data from 3 distinct US and European telestroke networks, we sought to externally validate the TeleStroke Mimic (TM) score in a broader population. METHODS: We evaluated the TM score in 1930 telestroke consults from the University of Utah, Georgia Regents University, and the German TeleMedical Project for Integrative Stroke Care Network. We report the area under the curve in receiver-operating characteristic curve analysis with 95% confidence interval for our previously derived TM score in which lower TM scores correspond with a higher likelihood of being a stroke mimic. RESULTS: Based on final diagnosis at the end of the telestroke consultation, there were 630 of 1930 (32.6%) stroke mimics in the external validation cohort. All 6 variables included in the score were significantly different between patients with ischemic cerebrovascular disease versus stroke mimics. The TM score performed well (area under curve, 0.72; 95% confidence interval, 0.70-0.73; P<0.001), similar to our prior external validation in the Partners National Telestroke Network. CONCLUSIONS: The TM score's ability to predict the presence of a stroke mimic during telestroke consultation in these diverse cohorts was similar to its performance in our original cohort. Predictive decision-support tools like the TM score may help highlight key clinical differences between mimics and patients with stroke during complex, time-critical telestroke evaluations.

Characterization of Biaxial Stretch as an In Vitro Model of Traumatic Brain Injury to the Blood-Brain Barrier.

Traumatic brain injury (TBI) is one of the major causes of disability in the USA. It occurs when external mechanical forces induce brain damage that causes deformation of brain tissue. TBI is also associated with alterations of the blood-brain barrier (BBB). Using primary rat brain microvascular endothelial cells as an in vitro BBB model, the effects of biaxial stretch were characterized at 5, 10, 15, 25, and 50% deformation using a commercially available system. The results were compared to the effects of mild and moderate TBI in vivo, induced by the weight-drop method in mice. In vitro, live/dead cells, lactate dehydrogenase (LDH) release, caspase 3/7 staining, and tight junction (TJ) protein expression were evaluated 24 h after a single stretch episode. In vivo, Evans blue extravasation, serum levels of S100ß, and TJ protein expression were evaluated. Stretch induced a deformation-dependent increase in LDH release, cell death, and activation of caspase 3/7, suggesting the induction of apoptosis. Interestingly, low magnitudes of deformation increased the expression of TJ proteins, likely in an attempt to compensate for stretch damage. High magnitudes of deformation decreased the expression of TJ proteins, suggesting that the damage was too severe to counteract. In vivo, mild TBI did not affect BBB permeability or the expression of TJ proteins. However, moderate TBI significantly increased BBB permeability and decreased the expression of these proteins, similar to the results obtained with a high magnitude deformation. These data support the use biaxial stretch as valuable tool in the study of TBI in vitro.

Isolation and Culture of Brain Microvascular Endothelial Cells for In Vitro Blood-Brain Barrier Studies.

The blood-brain barrier (BBB) is essential to maintain the proper microenvironment for brain function. Although formed by different cell types, the endothelial cells (ECs) of the brain microvessels provide the BBB with its selective permeability. To study the BBB in vitro, EC lines as well as primary isolated ECs have been used. In this chapter, we will provide a detailed protocol on how to isolate and culture primary brain microvascular endothelial cells from different species for use as in vitro models of the BBB. When performed properly, this protocol will allow one to obtain a pure culture of brain microvascular endothelial cells with which to analyze the effects of therapeutic and toxic agents on BBB functions.