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1.  Early Detection of Widespread Progressive Brain Injury after Cardiac Arrest: A Single Case DTI and Post-Mortem Histology Study 
PLoS ONE  2014;9(3):e92103.
We tested the hypothesis in sense of a proof of principle that white matter (WM) degeneration after cardiopulmonary arrest (CPA) can be assessed much earlier by diffusion tensor imaging (DTI) than by conventional MRI.
We performed DTI and T2-weighted FLAIR imaging over four serial acquisitions of a 76-year-old man with unresponsive wakefulness syndrome at day 41, 75, 173 and 284 after CPA. DTI was also performed in ten healthy control subjects. Fractional anisotropy (FA) derived from DTI was assessed in eleven regions of interest within the cerebral white matter (WM) and compared with post-mortem neuropathological findings.
In contrast to conventional FLAIR images that revealed only circumscribed WM damage, the first DTI demonstrated significant reduction of FA across the whole WM. The following FLAIR images (MRI 2-4) revealed increasing atrophy and leukoaraiosis paralleled by clinical deterioration with reduction of wakefulness and intractable seizures. Neuropathological findings confirmed the widespread and marked brain injury following CPA.
DTI may help to evaluate microstructural brain damage following CPA and may have predictive value for further evolution of cerebral degeneration in the chronic phase after CPA.
PMCID: PMC3954875  PMID: 24633135
2.  Continuous Cardiac Troponin I Release in Fabry Disease 
PLoS ONE  2014;9(3):e91757.
Fabry disease (FD) is a rare lysosomal storage disorder also affecting the heart. The aims of this study were to determine the frequency of cardiac troponin I (cTNI) elevation, a sensitive parameter reflecting myocardial damage, in a smaller cohort of FD-patients, and to analyze whether persistent cTNI can be a suitable biomarker to assess cardiac dysfunction in FD.
cTNI values were determined at least twice per year in 14 FD-patients (6 males and 8 females) regularly followed-up in our centre. The data were related to other parameters of heart function including cardiac magnetic resonance imaging (cMRI).
Three patients (21%) without specific vascular risk factors other than FD had persistent cTNI-elevations (range 0.05–0.71 ng/ml, normal: <0.01). cMRI disclosed late gadolinium enhancement (LGE) in all three individuals with cTNI values ≥0.01, while none of the 11 patients with cTNI <0.01 showed a pathological enhancement (p<0.01). Two subjects with increased cTNI-values underwent coronary angiography, excluding relevant stenoses. A myocardial biopsy performed in one during this procedure demonstrated substantial accumulation of globotriaosylceramide (Gb3) in cardiomyocytes.
Continuous cTNI elevation seems to occur in a substantial proportion of patients with FD. The high accordance with LGE, reflecting cardiac dysfunction, suggests that cTNI-elevation can be a useful laboratory parameter for assessing myocardial damage in FD.
PMCID: PMC3953535  PMID: 24626231
3.  Physical, Cognitive and Emotional Factors Contributing to Quality of Life, Functional Health and Participation in Community Dwelling in Chronic Kidney Disease 
PLoS ONE  2014;9(3):e91176.
Quality of life (QoL) impairment is a well-known consequence of chronic kidney disease (CKD). The factors influencing QoL and late life functional health are poorly examined.
Using questionnaires combined with neuropsychological examinations, we prospectively evaluated physical, cognitive, and emotional factors influencing QoL, functional health and participation in community dwelling in 119 patients with CKD stages 3–5 including hemodialysis (61.5±15.7years; 63% men) and 54 control patients of the same age without CKD but with similar cardiovascular risk profile.
Compared with control patients, CKD patients showed impairment of the physical component of QoL and overall function, assessed by the SF-36 and LLFDI, whereas disability, assessed by LLFDI, was selectively impaired in CKD patients on hemodialysis. Multivariable linear regressions (forced entry) confirmed earlier findings that CKD stage (β = −0.24; p = 0.012) and depression (β = −0.30; p = 0.009) predicted the QoL physical component. Hitherto unknown, CKD stage (β = −0.23; p = 0.007), cognition (β = 0.20; p = 0.018), and depression (β = −0.51; <0.001) predicted disability assessed by the LLFDI, while age (β = −0.20; p = 0.023), male gender (B = 5.01; p = 0.004), CKD stage (β = −0.23; p = 0.005), stroke history (B = −9.00; p = 0.034), and depression (β = −0.41; p<0.001) predicted overall function. Interestingly, functional health deficits, cognitive disturbances, depression, and anxiety were evident almost only in CKD patients with coronary heart disease (found in 34.2% of CKD patients). The physical component of QoL and functional health decreased with age and depressive symptoms, and increased with cognitive abilities.
In CKD, QoL, functional health, and participation in community dwelling are influenced by physical, cognitive, and emotional factors, most prominently in coronary heart disease patients.
PMCID: PMC3948783  PMID: 24614180
4.  Combined Acquisition Technique (CAT) for Neuroimaging of Multiple Sclerosis at Low Specific Absorption Rates (SAR) 
PLoS ONE  2014;9(3):e91030.
To compare a novel combined acquisition technique (CAT) of turbo-spin-echo (TSE) and echo-planar-imaging (EPI) with conventional TSE. CAT reduces the electromagnetic energy load transmitted for spin excitation. This radiofrequency (RF) burden is limited by the specific absorption rate (SAR) for patient safety. SAR limits restrict high-field MRI applications, in particular.
Material and Methods
The study was approved by the local Medical Ethics Committee. Written informed consent was obtained from all participants. T2- and PD-weighted brain images of n = 40 Multiple Sclerosis (MS) patients were acquired by CAT and TSE at 3 Tesla. Lesions were recorded by two blinded, board-certificated neuroradiologists. Diagnostic equivalence of CAT and TSE to detect MS lesions was evaluated along with their SAR, sound pressure level (SPL) and sensations of acoustic noise, heating, vibration and peripheral nerve stimulation.
Every MS lesion revealed on TSE was detected by CAT according to both raters (Cohen’s kappa of within-rater/across-CAT/TSE lesion detection κCAT = 1.00, at an inter-rater lesion detection agreement of κLES = 0.82). CAT reduced the SAR burden significantly compared to TSE (p<0.001). Mean SAR differences between TSE and CAT were 29.0 (±5.7) % for the T2-contrast and 32.7 (±21.9) % for the PD-contrast (expressed as percentages of the effective SAR limit of 3.2 W/kg for head examinations). Average SPL of CAT was no louder than during TSE. Sensations of CAT- vs. TSE-induced heating, noise and scanning vibrations did not differ.
T2−/PD-CAT is diagnostically equivalent to TSE for MS lesion detection yet substantially reduces the RF exposure. Such SAR reduction facilitates high-field MRI applications at 3 Tesla or above and corresponding protocol standardizations but CAT can also be used to scan faster, at higher resolution or with more slices. According to our data, CAT is no more uncomfortable than TSE scanning.
PMCID: PMC3946656  PMID: 24608106
5.  Glatiramer acetate does not protect from acute ischemic stroke in mice 
The role of the immune system in the pathophysiology of acute ischemic stroke is increasingly recognized. However, targeted treatment strategies to modulate immunological pathways in stroke are still lacking. Glatiramer acetate is a multifaceted immunomodulator approved for the treatment of relapsing-remitting multiple sclerosis. Experimental studies suggest that glatiramer acetate might also work in other neuroinflammatory or neurodegenerative diseases apart from multiple sclerosis.
We evaluated the efficacy of glatiramer acetate in a mouse model of brain ischemia/reperfusion injury. 60 min of transient middle cerebral artery occlusion was induced in male C57Bl/6 mice. Pretreatment with glatiramer acetate (3.5 mg/kg bodyweight) 30 min before the induction of stroke did not reduce lesion volumes or improve functional outcome on day 1.
Glatiramer acetate failed to protect from acute ischemic stroke in our hands. Further studies are needed to assess the true therapeutic potential of glatiramer acetate and related immunomodulators in brain ischemia.
PMCID: PMC3943273  PMID: 24576335
Glatiramer acetate; Stroke; Inflammation; Neurodegeneration
6.  Altered Cortical Swallowing Processing in Patients with Functional Dysphagia: A Preliminary Study 
PLoS ONE  2014;9(2):e89665.
Current neuroimaging research on functional disturbances provides growing evidence for objective neuronal correlates of allegedly psychogenic symptoms, thereby shifting the disease concept from a psychological towards a neurobiological model. Functional dysphagia is such a rare condition, whose pathogenetic mechanism is largely unknown. In the absence of any organic reason for a patient's persistent swallowing complaints, sensorimotor processing abnormalities involving central neural pathways constitute a potential etiology.
In this pilot study we measured cortical swallow-related activation in 5 patients diagnosed with functional dysphagia and a matched group of healthy subjects applying magnetoencephalography. Source localization of cortical activation was done with synthetic aperture magnetometry. To test for significant differences in cortical swallowing processing between groups, a non-parametric permutation test was afterwards performed on individual source localization maps.
Swallowing task performance was comparable between groups. In relation to control subjects, in whom activation was symmetrically distributed in rostro-medial parts of the sensorimotor cortices of both hemispheres, patients showed prominent activation of the right insula, dorsolateral prefrontal cortex and lateral premotor, motor as well as inferolateral parietal cortex. Furthermore, activation was markedly reduced in the left medial primary sensory cortex as well as right medial sensorimotor cortex and adjacent supplementary motor area (p<0.01).
Functional dysphagia - a condition with assumed normal brain function - seems to be associated with distinctive changes of the swallow-related cortical activation pattern. Alterations may reflect exaggerated activation of a widely distributed vigilance, self-monitoring and salience rating network that interferes with down-stream deglutition sensorimotor control.
PMCID: PMC3929717  PMID: 24586948
7.  Ischemia-induced cell depolarization: does the hyperpolarization-activated cation channel HCN2 affect the outcome after stroke in mice? 
Brain ischemia is known to include neuronal cell death and persisting neurological deficits. A lack of oxygen and glucose are considered to be key mediators of ischemic neurodegeneration while the exact mechanisms are yet unclear. In former studies the expression of two different two-pore domain potassium (K2P) channels (TASK1, TREK1) were shown to ameliorate neuronal damage due to cerebral ischemia. In neurons, TASK channels carrying hyperpolarizing K+ leak currents, and the pacemaker channel HCN2, carrying depolarizing Ih, stabilize the membrane potential by a mutual functional interaction. It is assumed that this ionic interplay between TASK and HCN2 channels enhances the resistance of neurons to insults accompanied by extracellular pH shifts.
In C57Bl/6 (wildtype, WT), hcn2+/+ and hcn2-/- mice we used an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of HCN2 in stroke formation. Subsequent analyses comprise behavioural tests and hcn2 gene expression assays.
After 60 min of tMCAO induction in WT mice, we collected tissue samples at 6, 12, and 24 h after reperfusion. In the infarcted neocortex, hcn2 expression analyses revealed a nominal peak of hcn2 expression 6 h after reperfusion with a tendency towards lower expression levels with longer reperfusion times. Hcn2 gene expression levels in infarcted basal ganglia did not change after 6 h and 12 h. Only at 24 h after reperfusion, hcn2 expression significantly decreases by ~55%. However, 30 min of tMCAO in hcn2-/- as well as hcn2+/+ littermates induced similar infarct volumes. Behavioural tests for global neurological function (Bederson score) and motor function/coordination (grip test) were performed at day 1 after surgery. Again, we found no differences between the groups.
Here, we hypothesized that the absence of HCN2, an important functional counter player of TASK channels, affects neuronal survival during stroke-induced tissue damage. However, together with a former study on TASK3 these results implicate that both TASK3 and HCN2 which were supposed to be neuroprotective due to their pH-dependency, do not influence ischemic neurodegeneration during stroke in the tMCAO model.
PMCID: PMC3879998  PMID: 24373160
9.  T2* “Susceptibility Vessel Sign” Demonstrates Clot Location and Length in Acute Ischemic Stroke 
PLoS ONE  2013;8(10):e76727.
The aim of our study was to evaluate, in acute ischemic stroke patients, the diagnostic accuracy of the MRI susceptibility vessel sign (SVS) against catheter angiography (DSA) for the detection of the clot and its value in predicting clot location and length.
Materials and Methods
We identified consecutive patients (2006–2012) admitted to our center, where 1.5 T MRI is systematically implemented as first-line diagnostic work-up, with: (1) pre-treatment 6-mm-thick multislice 2D T2* sequence; (2) delay from MRI-to-DSA <3 hrs; (3) no fibrinolysis between MRI and DSA. The location and length of SVS on T2* was independently assessed by three readers, and compared per patient, per artery and per segment, to DSA findings, obtained by two different readers. Clot length measured on T2* and DSA were compared using intra-class correlation coefficient (ICC), Bland & Altman test and Passing & Bablok regression analysis.
On DSA, a clot was present in 85 patients, in 126 of 1190 (10.6%) arteries and 175 of 1870 (9.4%) segments. Sensitivity of the SVS, as sensed by the used protocol at 1.5 T, was 81.1% (69 of 85 patients) and was higher in anterior (55 of 63, 87.3%), than in posterior circulation stroke (14 of 22, 63.6%, p=0.02). Sensitivity/specificity was 69.8/99.6% (per artery) and 76.6/99.7% (per segment). Positive (PPV) and negative predictive value (NPV) and accuracy were all >94%. Inter- and intra-observer ICC was excellent for clot length as measured on T2* (ĸ ≥0.97) and as measured on DSA (ĸ ≥0.94). Correlation between T2* and DSA for clot length was excellent (ICC: 0.88, 95%CI: 0.81–0.92; Bland & Altman: mean bias of 1.6% [95%CI: -4.7 to 7.8%], Passing & Bablok: 0.91).
SVS is a specific marker of clot location in the anterior and posterior circulation. Clot length greater than 6 mm can be reliably measured on T2*.
PMCID: PMC3795632  PMID: 24146915
10.  Diffusion- and Perfusion-Weighted Imaging in Acute Lacunar Infarction: Is There a Mismatch? 
PLoS ONE  2013;8(10):e77428.
Characterization of lacunar infarction (LI) by use of multimodal MRI including diffusion- and perfusion-weighted imaging (DWI, PWI) is difficult because of the small lesion size. Only a few studies evaluated PWI in LI and the results are inconsistent.
In 16 LI patients who underwent initial MRI within 6 hours after symptom onset and follow-up MRI within 1 week demographics, clinical presentation, and MRI findings were analyzed with special emphasis on DWI and PWI findings. Time to peak maps were classified as showing a normal perfusion pattern or areas of hypoperfusion which were further categorized in mismatch (PWI>DWI), inverse mismatch (PWI
Of the 16 patients (mean age 65.5±12.9 years), 14 (87.5%) were male. Clinical symptoms comprised dysarthria (50%), hemiparesis (81.3%), and hemihypaesthesia (18.8%). Intravenous thrombolysis was performed in 7 (43.8%) patients. Clinical improvement was observed in 12 patients (75 %), while 2 (12.5%) patients showed a deterioration and another 2 (12.5%) a stable course. Acute ischemic lesions (mean volume of 0.46±0.29 cm3) were located in the thalamus (n=8, 50%), internal capsule (n=4, 25%), corona Radiata (n=3, 18.8%) and the mesencephalon (n=1, 6.3%). Circumscribed hypoperfusion (mean volume 0.61±0.48 cm3) was evident in 10 (62.5%) patients. Of these, 3 patients demonstrated a match, 4 an inverse mismatch, and 3 a mismatch between DWI and PWI lesion. Mean CBF and CBV ratios were 0.65±0.28 and 0.84±0.41 respectively. Growth of DWI lesions was observed in 7 (43.8%) and reversal of DWI lesions in 3 (18.8%) patients.
MRI allows identification of different DWI and PWI patterns in LI, including growth and reversal of ischemic lesions. Consequently, it may serve for a better characterization of this stroke subtype and support treatment decisions in daily clinical practice.
PMCID: PMC3795042  PMID: 24130885
PLoS ONE  2013;8(8):e71894.
Fabry disease is a rare X-linked inherited lysosomal storage disorder affecting multiple organ systems. It includes central nervous system involvement via micro- and macroangiopathic cerebral changes. Due to its clinical symptoms and frequent MRI lesions, Fabry disease is commonly misdiagnosed as multiple sclerosis. We present an overview of cases from Fabry centres in Germany initially misdiagnosed with multiple sclerosis and report the clinical, MR-tomographical, and laboratory findings.
Eleven Fabry patients (one male, ten females) initially diagnosed with multiple sclerosis were identified from 187 patient records (5.9%) and analyzed for presenting symptoms, results of the initial diagnostic workup, and the clinical course of the disease.
Four patients were identified as having a “possible” history of MS, and 7 patients as “definite” cases of multiple sclerosis (revised McDonald criteria). On average, Fabry disease was diagnosed 8.2 years (±9.8 years) after the MS diagnosis, and 12.8 years after onset of first symptoms (±10.3 years). All patients revealed white matter lesions on MRI. The lesion pattern and results of cerebrospinal fluid examination were inconsistent and non-specific. White matter lesion volumes ranged from 8.9 mL to 34.8 mL (mean 17.8 mL±11.4 mL). There was no association between extra-neurological manifestations or enzyme activity and lesion load.
There are several anamnestic and clinical hints indicating when Fabry disease should be considered a relevant differential diagnosis of multiple sclerosis, e.g. female patients with asymmetric, confluent white matter lesions on MRI, normal spinal MR imaging, ectatic vertebrobasilar arteries, proteinuria, or lack of intrathecally derived immunoglobulin synthesis.
PMCID: PMC3756019  PMID: 24015197
PLoS ONE  2013;8(8):e71132.
Current guidelines recommend withholding antithrombotic therapy (ATT) for at least 24 h in patients with acute ischemic stroke treated with thrombolytic therapy. Herein, we report a retrospective analysis of a single-centre experience on the safety and efficacy of antithrombotic therapy (ATT) started before or after 24 h of intravenous thrombolysis in a cohort of acute ischemic stroke patients.
A total of 139 patients (Rapid ATT group) received antithrombotic therapy before 24 h of thrombolysis, and 33 patients (Standard ATT group) after 24 h. The brain parenchyma and vessel status were assessed using simple CT scan on admission, multimodal CT scan at the end of thrombolysis, and angio-CT/MRI scan at day 3. Functional outcome was scored using the modified Rankin Scale (mRS) at day 90.
The two ATT groups had similar demographics, stroke subtypes, baseline NIHSS, thrombolytic strategies, vessel-patency rates at the end of thrombolysis, and incidence of bleeding complications at follow up. At day 3, the Rapid ATT group had a non-significant improved vessel-patency rate than the Standard ATT group. At day 90, a greater proportion of patients in the rapid ATT group had shifted down the mRS, and had improved in the NIHSS score.
ATT initiated before 24 h of intravenous thrombolytic therapy in acute stroke patients disclosed no safety concerns compared with a conventional antithrombotic therapy delay of 24 h and showed better functional outcome at follow up. The value of early initiation of ATT after thrombolysis deserves further assessment in randomized controlled trials.
PMCID: PMC3738638  PMID: 23951093
PLoS ONE  2013;8(8):e69529.
Inflammation is a pathophysiological hallmark of many diseases of the brain. Specific imaging of cells and molecules that contribute to cerebral inflammation is therefore highly desirable, both for research and in clinical application. The 18 kDa translocator protein (TSPO) has been established as a suitable target for the detection of activated microglia/macrophages. A number of novel TSPO ligands have been developed recently. Here, we evaluated the high affinity TSPO ligand DPA-714 as a marker of brain inflammation in two independent animal models. For the first time, the specificity of radiolabeled DPA-714 for activated microglia/macrophages was studied in a rat model of epilepsy (induced using Kainic acid) and in a mouse model of stroke (transient middle cerebral artery occlusion, tMCAO) using high-resolution autoradiography and immunohistochemistry. Additionally, cold-compound blocking experiments were performed and changes in blood-brain barrier (BBB) permeability were determined. Target-to-background ratios of 2 and 3 were achieved in lesioned vs. unaffected brain tissue in the epilepsy and tMCAO models, respectively. In both models, ligand uptake into the lesion corresponded well with the extent of Ox42- or Iba1-immunoreactive activated microglia/macrophages. In the epilepsy model, ligand uptake was almost completely blocked by pre-injection of DPA-714 and FEDAA1106, another high-affinity TSPO ligand. Ligand uptake was independent of the degree of BBB opening and lesion size in the stroke model. We provide further strong evidence that DPA-714 is a specific ligand to image activated microglia/macrophages in experimental models of brain inflammation.
PMCID: PMC3732268  PMID: 23936336
PLoS ONE  2013;8(7):e70124.
The Sphingosine-1-phosphate (S1P) signaling pathway is known to influence pathophysiological processes within the brain and the synthetic S1P analog FTY720 has been shown to provide neuroprotection in experimental models of acute stroke. However, the effects of a manipulation of S1P signaling at later time points after experimental stroke have not yet been investigated. We examined whether a relatively late initiation of a FTY720 treatment has a positive effect on long-term neurological outcome with a focus on reactive astrogliosis, synapses and neurotrophic factors.
We induced photothrombotic stroke (PT) in adult C57BL/6J mice and allowed them to recover for three days. Starting on post-stroke day 3, mice were treated with FTY720 (1 mg/kg b.i.d.) for 5 days. Behavioral outcome was observed until day 31 after photothrombosis and periinfarct cortical tissue was analyzed using tandem mass-spectrometry, TaqMan®analysis and immunofluorescence.
FTY720 treatment results in a significantly better functional outcome persisting up to day 31 after PT. This is accompanied by a significant decrease in reactive astrogliosis and larger post-synaptic densities as well as changes in the expression of vascular endothelial growth factor α (VEGF α). Within the periinfarct cortex, S1P is significantly increased compared to healthy brain tissue.
Besides its known neuroprotective effects in the acute phase of experimental stroke, the initiation of FTY720 treatment in the convalescence period has a positive impact on long-term functional outcome, probably mediated through reduced astrogliosis, a modulation in synaptic morphology and an increased expression of neurotrophic factors.
PMCID: PMC3729514  PMID: 23936150
PLoS ONE  2013;8(7):e66960.
We examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice.
Several antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding.
Platelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery.
Thrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept IV, compared to Fc only. 1 mg/kg Revacept IV applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen.
Revacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.
PMCID: PMC3720811  PMID: 23935828
Neurobiology of disease  2008;33(1):1-11.
Oxygen depletion (O2) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42 ± 2%) and O2 deprivation (36 ± 5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1−/− mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.
PMCID: PMC3714864  PMID: 18930826
cerebral ischemia; transient middle cerebral artery occlusion; Two-pore domain potassium channels; TASK channels; thalamic neurons; TASK1−/− mice; electrophysiology
PLoS ONE  2013;8(7):e68996.
Recent studies have suggested a protective role of physiological β-amyloid autoantibodies (Aβ-autoantibodies) in Alzheimer’s disease (AD). However, the determination of both free and dissociated Aβ-autoantibodies in serum hitherto has yielded inconsistent results regarding their function and possible biomarker value. Here we report the application of a new sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of antigen-bound Aβ-autoantibodies (intact Aβ-IgG immune complexes) in serum and cerebrospinal fluid (CSF) of a total number of 112 AD patients and age- and gender-matched control subjects. Both serum and CSF levels of Aβ-IgG immune complexes were found to be significantly higher in AD patients compared to control subjects. Moreover, the levels of Aβ-IgG complexes were negatively correlated with the cognitive status across the groups, increasing with declining cognitive test performance of the subjects. Our results suggest a contribution of IgG-type autoantibodies to Aβ clearance in vivo and an increased immune response in AD, which may be associated with deficient Aβ-IgG removal. These findings may contribute to elucidating the role of Aβ-autoantibodies in AD pathophysiology and their potential application in AD diagnosis.
PMCID: PMC3715516  PMID: 23874844
Science signaling  2010;3(103):ra1.
Platelet aggregation is essential for hemostasis, but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is increased function of the major adhesive receptor, αIIbβ3 integrin, which enables adhesion and aggregation. Phospholipases (PL), in response to agonist receptor stimulation, cleave membrane phospholipids to generate lipid second messengers. An essential role in platelet activation has been established for PLC, but not for PLD and its product phosphatidic acid. Here, we report the generation of Pld1−/− mice and show that their platelets display impaired αIIbβ3 integrin activation in response to classic agonists, and defective glycoprotein Ib-dependent aggregate formation under high shear flow conditions. This defect resulted in protection from thrombosis and ischemic brain infarction, without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.
PMCID: PMC3701458  PMID: 20051593
PLoS ONE  2013;8(7):e69252.
δ-opioid receptor (DOR) activation reduced brain ischemic infarction and attenuated neurological deficits, while DOR inhibition aggravated the ischemic damage. The underlying mechanisms are, however, not well understood yet. In this work, we asked if DOR activation protects the brain against ischemic injury through a brain-derived neurotrophic factor (BDNF) -TrkB pathway.
We exposed adult male Sprague-Dawley rats to focal cerebral ischemia, which was induced by middle cerebral artery occlusion (MCAO). DOR agonist TAN-67 (60 nmol), antagonist Naltrindole (100 nmol) or artificial cerebral spinal fluid was injected into the lateral cerebroventricle 30 min before MCAO. Besides the detection of ischemic injury, the expression of BDNF, full-length and truncated TrkB, total CREB, p-CREB, p-ATF and CD11b was detected by Western blot and fluorescence immunostaining.
DOR activation with TAN-67 significantly reduced the ischemic volume and largely reversed the decrease in full-length TrkB protein expression in the ischemic cortex and striatum without any appreciable change in cerebral blood flow, while the DOR antagonist Naltrindole aggregated the ischemic injury. However, the level of BDNF remained unchanged in the cortex, striatum and hippocampus at 24 hours after MCAO and did not change in response to DOR activation or inhibition. MCAO decreased both total CREB and pCREB in the striatum, but not in the cortex, while DOR inhibition promoted a further decrease in total and phosphorylated CREB in the striatum and decreased pATF-1 expression in the cortex. In addition, MCAO increased C11b expression in the cortex, striatum and hippocampus, and DOR activation specifically attenuated the ischemic increase in the cortex but not in the striatum and hippocampus.
DOR activation rescues TrkB signaling by reversing ischemia/reperfusion induced decrease in the full-length TrkB receptor and reduces brain injury in ischemia/reperfusion
PMCID: PMC3699518  PMID: 23844255
PLoS ONE  2013;8(6):e67063.
The inflammatory response following ischemic stroke is dominated by innate immune cells: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.
PMCID: PMC3692438  PMID: 23825621
Human cerebral small vessel disease (CSVD) has distinct histopathologic and imaging findings in its advanced stages. In spontaneously hypertensive stroke-prone rats (SHRSP), a well-established animal model of CSVD, we recently demonstrated that cerebral microangiopathy is initiated by early microvascular dysfunction leading to the breakdown of the blood–brain barrier and an activated coagulatory state resulting in capillary and arteriolar erythrocyte accumulations (stases). In the present study, we investigated whether initial microvascular dysfunction and other stages of the pathologic CSVD cascade can be detected by serial magnetic resonance imaging (MRI).
Fourteen SHRSP and three control (Wistar) rats (aged 26–44 weeks) were investigated biweekly by 3.0 Tesla (3 T) MRI. After perfusion, brains were stained with hematoxylin–eosin and histology was correlated with MRI data. Three SHRSP developed terminal CSVD stages including cortical, hippocampal, and striatal infarcts and macrohemorrhages, which could be detected consistently by MRI. Corresponding histology showed small vessel thromboses and increased numbers of small perivascular bleeds in the infarcted areas. However, 3 T MRI failed to visualize intravascular erythrocyte accumulations, even in those brain regions with the highest densities of affected vessels and the largest vessels affected by stases, as well as failing to detect small perivascular bleeds.
Serial MRI at a field strength of 3 T failed to detect the initial microvascular dysfunction and subsequent small perivascular bleeds in SHRSP; only terminal stages of cerebral microangiopathy were reliably detected. Further investigations at higher magnetic field strengths (7 T) using blood- and flow-sensitive sequences are currently underway.
PMCID: PMC3724477  PMID: 23800299
Cerebral small vessel disease; SHRSP; MRI
PLoS ONE  2013;8(6):e66657.
Pannexin1 (Panx1) is a plasma membrane channel permeable to relatively large molecules, such as ATP. In the central nervous system (CNS) Panx1 is found in neurons and glia and in the immune system in macrophages and T-cells. We tested the hypothesis that Panx1-mediated ATP release contributes to expression of Experimental Autoimmune Encephalomyelitis (EAE), an animal model for multiple sclerosis, using wild-type (WT) and Panx1 knockout (KO) mice. Panx1 KO mice displayed a delayed onset of clinical signs of EAE and decreased mortality compared to WT mice, but developed as severe symptoms as the surviving WT mice. Spinal cord inflammatory lesions were also reduced in Panx1 KO EAE mice during acute disease. Additionally, pharmacologic inhibition of Panx1 channels with mefloquine (MFQ) reduced severity of acute and chronic EAE when administered before or after onset of clinical signs. ATP release and YoPro uptake were significantly increased in WT mice with EAE as compared to WT non-EAE and reduced in tissues of EAE Panx1 KO mice. Interestingly, we found that the P2X7 receptor was upregulated in the chronic phase of EAE in both WT and Panx1 KO spinal cords. Such increase in receptor expression is likely to counterbalance the decrease in ATP release recorded from Panx1 KO mice and thus contribute to the development of EAE symptoms in these mice. The present study shows that a Panx1 dependent mechanism (ATP release and/or inflammasome activation) contributes to disease progression, and that inhibition of Panx1 using pharmacology or gene disruption delays and attenuates clinical signs of EAE.
PMCID: PMC3688586  PMID: 23885286
PLoS ONE  2013;8(6):e59274.
Post-stroke hypoxia is common, and may adversely affect outcome. We have recently shown that oxygen supplementation may improve early neurological recovery. Here, we report the six-month outcomes of this pilot study.
Patients with a clinical diagnosis of acute stroke were randomized within 24 h of admission to oxygen supplementation at 2 or 3 L/min for 72 h or to control treatment (room air). Outcomes (see below) were assessed by postal questionnaire at 6 months. Analysis was by intention-to-treat, and statistical significance was set at p≤0.05.
Out of 301 patients randomized two refused/withdrew consent and 289 (148 in the oxygen and 141 in the control group) were included in the analysis: males 44%, 51%; mean (SD) age 73 (12), 71 (12); median (IQR) National Institutes of Health Stroke Scale score 6 (3, 10), 5 (3, 10) for the two groups respectively. At six months 22 (15%) patients in the oxygen group and 20 (14%) in the control group had died; mean survival in both groups was 162 days (p = 0.99). Median (IQR) scores for the primary outcome, the modified Rankin Scale, were 3 (1, 5) and 3 (1, 4) for the oxygen and control groups respectively. The covariate-adjusted odds ratio was 1.04 (95% CI 0.67, 1.60), indicating that the odds of a lower (i.e. better) score were non-significantly higher in the oxygen group (p = 0.86). The mean differences in the ability to perform basic (Barthel Index) and extended activities of daily living (NEADL), and quality of life (EuroQol) were also non-significant.
None of the key outcomes differed at 6 months between the groups. Although not statistically significant and generally of small magnitude, the effects were predominantly in favour of the oxygen group; a larger trial, powered to show differences in longer-term functional outcomes, is now on-going.
Trial Registration ISRCTN12362720; 2004-001866-41
PMCID: PMC3670882  PMID: 23755093
PLoS ONE  2013;8(5):e63810.
It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s−1) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf −/− mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf −/− and Vwf +/+ blood, exposed to VWF at 1800 s−1 in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf−/− mice than from Vwf+/+ mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf−/− mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s−1 on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.
PMCID: PMC3667798  PMID: 23737952

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