In recent years, argon has been shown to exert neuroprotective effects in an array of models. However, the mechanisms by which argon exerts its neuroprotective characteristics remain unclear. ...Accumulating evidence imply that argon may exert neuroprotective effects via modulating the activation and polarization of microglia/macrophages after ischemic stroke. In the present study, we analyzed the underlying neuroprotective effects of delayed argon application until 7 days after reperfusion and explored the potential mechanisms.
Twenty-one male Wistar rats underwent transient middle cerebral artery occlusion or sham surgery randomly for 2 h using the endoluminal thread model. Three hours after transient middle cerebral artery occlusion induction and 1 h after reperfusion, animals received either 50% vol Argon/50% vol O
or 50% vol N
/50% vol O
for 1 h. The primary outcome was the 6-point neuroscore from 24 h to d7 after reperfusion. Histological analyses including infarct volume, survival of neurons (NeuN) at the ischemic boundary zone, white matter integrity (Luxol Fast Blue), microglia/macrophage activation (Iba1), and polarization (Iba1/Arginase1 double staining) on d7 were conducted as well. Sample size calculation was performed using nQuery Advisor + nTerim 4.0. Independent t test, one-way ANOVA and repeated measures ANOVA were performed, respectively, for statistical analysis (SPSS 23.0).
The 6-point neuroscore from 24 h to d7 after reperfusion showed that tMCAO Ar group displayed significantly improved neurological performance compared to tMCAO N
group (p = 0.026). The relative numbers of NeuN-positive cells in the ROIs of tMCAO Ar group significantly increased compared to tMCAO N
group (p = 0.010 for cortex and p = 0.011 for subcortex). Argon significantly suppressed the microglia/macrophage activation as revealed by Iba1 staining (p = 0.0076) and promoted the M2 microglia/macrophage polarization as revealed by Iba1/Arginase 1 double staining (p = 0.000095).
Argon administration with a 3 h delay after stroke onset and 1 h after reperfusion significantly alleviated neurological deficit within the first week and preserved the neurons at the ischemic boundary zone 7 days after stroke. Moreover, argon reduced the excessive microglia/macrophage activation and promoted the switch of microglia/macrophage polarization towards the anti-inflammatory M2 phenotype. Studies making efforts to further elucidate the protective mechanisms and to benefit the translational application are of great value.
The assessment of treatment response in glioblastoma is difficult with MRI because reactive blood-brain barrier alterations with contrast enhancement can mimic tumor progression. In this study, we ...investigated the predictive value of PET using O-(2-(18)F-fluoroethyl)-l-tyrosine ((18)F-FET PET) during treatment.
In a prospective study, 25 patients with glioblastoma were investigated by MRI and (18)F-FET PET after surgery (MRI-/FET-1), early (7-10 d) after completion of radiochemotherapy with temozolomide (RCX) (MRI-/FET-2), and 6-8 wk later (MRI-/FET-3). Maximum and mean tumor-to-brain ratios (TBR(max) and TBR(mean), respectively) were determined by region-of-interest analyses. Furthermore, gadolinium contrast-enhancement volumes on MRI (Gd-volume) and tumor volumes in (18)F-FET PET images with a tumor-to-brain ratio greater than 1.6 (T(vol 1.6)) were calculated using threshold-based volume-of-interest analyses. The patients were grouped into responders and nonresponders according to the changes of these parameters at different cutoffs, and the influence on progression-free survival and overall survival was tested using univariate and multivariate survival analyses and by receiver-operating-characteristic analyses.
Early after completion of RCX, a decrease of both TBR(max) and TBR(mean) was a highly significant and independent statistical predictor for progression-free survival and overall survival. Receiver-operating-characteristic analysis showed that a decrease of the TBR(max) between FET-1 and FET-2 of more than 20% predicted favorable survival corrected, with a sensitivity of 83% and a specificity of 67% (area under the curve, 0.75). Six to eight weeks later, the predictive value of TBR(max) and TBR(mean) was less significant, but an association between a decrease of T(vol 1.6) and PFS was noted. In contrast, Gd-volume changes had no significant predictive value for survival.
In contrast to Gd-volumes on MRI, changes in (18)F-FET PET may be a valuable parameter to assess treatment response in glioblastoma and to predict survival time.
Minipigs are frequently used in (neuro-)interventional research. Longitudinal experiments may require repeated vessel access via the femoral artery. Anticoagulation and incompliance of the animals ...necessitates the use of a vascular closure device (VCD). The effects of the Angio-Seal VCD in minipigs were longitudinally assessed. Minipig (42±8.4 kg body weight) femoral arteries were sealed using the 8F (n = 6) or 6F (n = 7) Angio-Seal VCD. The pre-interventional femoral artery diameter was 5.1±0.4 mm (4.3-5.8 mm). Sealed puncture sites were analysed angiographically as well as by computed tomography angiography (CTA) for a mean period of 14.1±8.0 weeks (1-22 weeks). All animals were constantly treated with acetylsalicylic acid (ASS) (450 mg/d (n = 7) or 100 mg/d (n = 1)) and clopidogrel (75 mg/d (n = 8)). Non-instrumented (n = 2) and arteries sealed using the VCD (n = 2) were examined histologically. No postoperative hemorrhagic complications were observed. Three arteries were occluded after VCD placement (1 animal diagnosed after 4 weeks (8F), 2 animals after 1 week (6F)) and remained so until the end of the experiments after 22, 12 and 4 weeks, respectively. In one artery a 50% stenosis 8 weeks after application of a 6F Angio-Seal was detected. In 69.2% (n = 9) the VCD was applied without complications. Histopathological analysis of the sealed arterial segments showed subtotal obliteration of the vessel lumen, formation of collagenous tissue and partial damage of the internal elastic lamina. The Angio-Seal VCD prevents relevant hemorrhagic complications in minipigs treated with dual platelet inhibition, but is associated with increased vessel occlusion rates.
Delayed cell death in the penumbra region of acute ischemic stroke occurs through apoptotic mechanisms, making it amenable to therapeutic interventions. Fas/CD95 mediates apoptotic cell death in ...response to external stimuli. In mature neurons, Fas/CD95 signaling is modulated by Fas‐apoptotic inhibitory molecule 2 (Faim2), which reduces cell death in animal models of stroke, meningitis, and Parkinson disease. Erythropoietin (EPO) has been studied as a therapeutic strategy in ischemic stroke. Erythropoietin stimulates the phosphatidylinositol‐3 kinase/Akt (PI3K/Akt) pathway, which regulates Faim2 expression. Therefore, up‐regulation of Faim2 may contribute to neuroprotection by EPO. Male Faim2‐deficient mice (Faim2−/−) and wild‐type littermates (WT) were subjected to 30 min of middle cerebral artery occlusion (MCAo) followed by 72 h of reperfusion. EPO was applied before (30 min) and after (24 and 48 h) MCAo. In WT mice application of EPO at a low dose (5000 U/kg) significantly reduced stroke volume, whereas treatment with high dose (90 000 U/kg) did not. In Faim2−/− animals administration of low‐dose EPO did not result in a significant reduction in stroke volume. Faim2 expression as measured by quantitative reverse transcription polymerase chain reaction (qRT‐PCR) increased after low‐dose EPO but not with high dose. An extensive phenotyping including analysis of cerebral vessel architecture did not reveal confounding differences between the genotypes. In human post‐mortem brain Faim2 displayed a differential expression in areas of penumbral ischemia. Faim2 up‐regulation may contribute to the neuroprotective effects of low‐dose erythropoietin in transient brain ischemia. The dose‐dependency may explain mixed effects of erythropoietin observed in clinical stroke trials.
Delayed cell death in the penumbra region of acute ischemic stroke occurs through apoptotic mechanisms, making it amenable to therapeutic interventions. The signaling pathway by which erythropoietin (EPO) protects neurons in cerebral ischemia has not been resolved. We hypothesize that Fas‐apoptotic inhibitory molecule 2 (Faim2) is involved in EPO‐mediated neuroprotection, in a pathway that also involves blockade of the death receptor Fas, also known as apoptosis antigen 1. We tested our hypothesis in an animal model of transient cerebral ischemia. Results from this study revealed that EPO reduces ischemia‐related cell death in mice but failed in clinical stroke studies. Fas/CD95 signaling contributes to ischemia‐related cell death and can be inhibited by Faim2. Mice lacking Faim2 were not protected by EPO doses that reduced ischemia‐related cell death in wild type mice. We found up‐regulated Faim2 after EPO and suggest that Faim2 may contribute to EPO‐mediated neuroprotection after stroke. FADD, Fas‐associated death domain‐containing protein.
OBJECTIVE:To analyze antisynthetase syndrome–associated myositis by modern myopathologic methods and to define its place in the spectrum of idiopathic inflammatory myopathies (IIMs).
METHODS:Skeletal ...muscle biopsies from antisynthetase syndrome–associated myositis and other IIMs from different institutions worldwide were analyzed by histopathology, quantitative PCR, and electron microscopy.
RESULTS:Myonuclear actin filament inclusions were identified as a unique morphologic hallmark of antisynthetase syndrome–associated myositis. Nuclear actin inclusions were never found in dermatomyositis, polymyositis, sporadic inclusion body myositis, autoimmune necrotizing myopathy associated with signal recognition particle or 3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies, or nonspecific myositis associated with other systemic diseases, harboring myositis-associated autoantibodies, and presenting myofiber necrosis. We show that molecules involved in actin filament formation and actin shuttling mechanisms are altered in antisynthetase syndrome, and may thus be involved in pathologic myonuclear actin aggregation. In addition, we have identified a typical topographic distribution of necrotic myofibers predominantly located at the periphery of muscle fascicles accompanied by inflammation and destruction of the perimysial connective tissue.
CONCLUSION:Antisynthetase syndrome–associated myositis is characterized by distinctive myonuclear actin filament inclusions, including rod formations and a typical necrotizing perimysial myositis. This supports the hypothesis that antisynthetase syndrome–associated myositis is unique and should not be grouped among dermatomyositis, polymyositis, sporadic inclusion body myositis, necrotizing autoimmune myositis, or nonspecific myositis.
CLASSIFICATION OF EVIDENCE:This study provides Class II evidence that for patients with IIMs, the presence of myonuclear actin filament inclusions accurately identifies patients with antisynthetase syndrome–associated myositis (sensitivity 81%, specificity 100%).
The liver-derived plasma protein fetuin-A is strongly expressed during fetal life, hence its name. Fetuin-A protein is normally present in most fetal organs and tissues, including brain tissue. ...Fetuin-A was neuroprotective in animal models of cerebral ischemia and lethal chronic inflammation, suggesting a role beyond the neonatal period. Little is known, however, on the presence of fetuin-A in mature human brain tissue under different physiological and pathological conditions.
We studied by immunohistochemistry (IHC) the distribution of fetuin-A protein in mature human brain autopsy tissues from patients without neurological disease, patients with inflammatory brain disorders, and patients with ischemic brain lesions. To identify fetuin-A-positive cells in these tissues we co-localized fetuin-A with GFAP (astrocytes) and CD68 (macrophages, activated microglia).
Unlike previous reports, we detected fetuin-A protein also in mature human brain as would be expected from an abundant plasma protein also present in cerebrospinal fluid. Fetuin-A immunoreactivity was increased in ischemic white matter and decreased in inflamed cerebellar tissue. Fetuin-A immunostaining was predominantly associated with neurons and astrocytes. Unlike the developing brain, the adult brain lacked fetuin-A immunostaining in CD68-positive microglia. Our findings suggest a role for fetuin-A in tissue remodeling of neonatal brain, which becomes obsolete in the adult brain, but is re-activated in damaged brain tissue. To further assess the role of fetuin-A in the mature brain, animal models involving ischemia and inflammation need to be studied.
In a previous study from our group, argon has shown to significantly attenuate brain injury, reduce brain inflammation and enhance M
microglia/macrophage polarization until 7 days after ischemic ...stroke. However, the long-term effects of argon have not been reported thus far. In the present study, we analyzed the underlying neuroprotective effects and potential mechanisms of argon, up to 30 days after ischemic stroke. Argon administration with a 3 h delay after stroke onset and 1 h after reperfusion demonstrated long-term neuroprotective effect by preserving the neurons at the ischemic boundary zone 30 days after stroke. Furthermore, the excessive microglia/macrophage activation in rat brain was reduced by argon treatment 30 days after ischemic insult. However, long-lasting neurological improvement was not detectable. More sensorimotor functional measures, age- and disease-related models, as well as further histological and molecular biological analyses will be needed to extend the understanding of argon's neuroprotective effects and mechanism of action after ischemic stroke.
Recently aside from the "classic" endovascular monofilament perforation technique to induce experimental subarachnoid hemorrhage (SAH) a modification using a tungsten wire advanced through a guide ...tube has been described. We aim to assess both techniques for their success rate (induction of SAH without confounding pathologies) as primary endpoint. Further, the early tissue lesion pattern as evidence for early brain injury will be analyzed as secondary endpoint. Sprague Dawley rats (n=39) were randomly assigned to receive either Sham surgery (n=4), SAH using the "classic" technique (n=18) or using a modified technique (n=17). Course of intracranial pressure (ICP) and regional cerebral blood flow (rCBF) was analyzed; subsequent pathologies were documented either 6 or 24 h after SAH. Hippocampal tissue samples were analyzed via immunohistochemistry and western blotting. SAH-induction, regardless of confounding pathologies, was independent from type of technique (p=0.679). There was no significant difference concerning case fatality rate (classic: 40%; modified: 20%; p=0.213). Successful induction of SAH without collateral ICH or SDH was possible in 40% with the classic and in 86.7% with the modified technique (p=0.008). Peak ICP levels differed significantly between the two groups (classic: 94 +/- 23 mmHg; modified: 68 +/- 19 mmHg; p=0.003). Evidence of early cellular stress response and activation of apoptotic pathways 6 h after SAH was demonstrated. The extent of stress response is not dependent on type of technique. Both tested techniques successfully produce SAH including activation of an early stress response and apoptotic pathways in the hippocampal tissue. However, the induction of SAH with less confounding pathologies was more frequently achieved with the modified tungsten wire technique.
OBJECTIVE:Despite the introduction of mild therapeutic hypothermia into postcardiac arrest care, cerebral and myocardial injuries represent the limiting factors for survival after cardiac arrest. ...Administering xenon may confer an additional neuroprotective effect after successful cardiopulmonary resuscitation due to its ability to stabilize cellular calcium homeostasis via N-methyl-D-aspartate-receptor antagonism.
DESIGN:In a porcine model, we evaluated effects of xenon treatment in addition to therapeutic hypothermia on neuropathologic and functional outcomes after cardiopulmonary resuscitation.
SETTING:Prospective, randomized, laboratory animal study.
SUBJECTS:Fifteen male pigs.
INTERVENTIONS:Following 10 mins of cardiac arrest and 6 mins of cardiopulmonary resuscitation, ten pigs were randomized to receive either mild therapeutic hypothermia (33°C for 16 hrs) or mild therapeutic hypothermia 1 xenon (70% for 1 hr). Five animals served as normothermic controls.
MEASUREMENTS AND MAIN RESULTS:Gross hemodynamic variables were measured using right-heart catheterization. Neurocognitive performance was evaluated for 5 days after cardiopulmonary resuscitation using a neurologic deficit score before the brains were harvested for histopathological analysis. All animals survived the observation period in the mild therapeutic hypothermia 1 xenon group while one animal in each of the other two groups died. Mild therapeutic hypothermia 1 xenon preserved cardiac output during the induction of mild therapeutic hypothermia significantly better than did mild therapeutic hypothermia alone (4.6 6 0.6 L/min vs. 3.2 6 1.6 L/min, p # .05). Both treatment groups showed significantly fewer necrotic lesions in the cerebral cortex, caudate nucleus, putamen, and in hippocampal sectors CA1 and CA3/4. However, only the combination of mild therapeutic hypothermia and xenon resulted in reduced astrogliosis in the CA1 sector and diminished microgliosis and perivascular inflammation in the putamen. Clinically, only the mild therapeutic hypothermia 1 xenon-treated animals showed significantly improved neurologic deficit scores over time (day 1 = 59.0 6 27.0 vs. day 5 = 4.0 6 5.5, p ø .05) as well as in comparison to the untreated controls on days 3 through 5 after cardiopulmonary resuscitation.
CONCLUSIONS:These results demonstrate that even a short exposure to xenon during induction of mild therapeutic hypothermia results in significant improvements in functional recovery and ameliorated myocardial dysfunction. (Crit Care Med 2012; 40:–1303)
TCF4 acts as a transcription factor that binds to the immunoglobulin enhancer Mu-E5/KE5 motif. Dominant variants in TCF4 are associated with the manifestation of Pitt-Hopkins syndrome, a rare disease ...characterized by severe mental retardation, certain features of facial dysmorphism and, in many cases, with abnormalities in respiratory rhythm (episodes of paroxysmal tachypnea and hyperventilation, followed by apnea and cyanosis). Frequently, patients also develop epilepsy, microcephaly, and postnatal short stature. Although TCF4 is expressed in skeletal muscle and TCF4 seems to play a role in myogenesis as demonstrated in mice, potential myopathological findings taking place upon the presence of dominant TCF4 variants are thus far not described in human skeletal muscle. To address the pathological effect of a novel deletion affecting exons 15 and 16 of TCF4 on skeletal muscle, histological and immunofluorescence studies were carried out on a quadriceps biopsy in addition to targeted transcript studies and global proteomic profiling. We report on muscle biopsy findings from a Pitt-Hopkins patient with a novel heterozygous deletion spanning exon 15 and 16 presenting with neuromuscular symptoms. Microscopic characterization of the muscle biopsy revealed moderate fiber type I predominance, imbalance in the proportion of fibroblasts co-expressing Vimentin and CD90, and indicate activation of the complement cascade in TCF4-mutant muscle. Protein dysregulations were unraveled by proteomic profiling. Transcript studies confirmed a mitochondrial vulnerability in muscle and confirmed reduced TCF4 expression. Our combined findings, for the first time, unveil myopathological changes as phenotypical association of Pitt-Hopkins syndrome and thus expand the current clinical knowledge of the disease as well as support data obtained on skeletal muscle of a mouse model.
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