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The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 µg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.β-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. [ABSTRACT FROM AUTHOR]

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain. [ABSTRACT FROM AUTHOR]

Abstract: Evaluation of functional outcome over the course of several weeks after ischemia is a key component in improving the clinical relevance of experimental stroke studies. Using a battery of behavioral tests, we characterized functional outcome in mice over 4 weeks following 30min of proximal middle cerebral artery occlusion (MCAo). We evaluated rotarod, chimney, pole and cylinder tests to assess short term functional deficits in a transient stroke model which induces infarcts mainly in the striatum. The corner test, adhesive removal test, cylinder test, catwalk, paw preference test and novel tests of rotation were evaluated for long-term functional outcome. Rotarod detected deficits within the first week and pole test was reliable up to intermediate time points after MCAo. Corner test, adhesive removal test, catwalk and paw preference test detected deficits for up to 4 weeks, as did the novel corner rotation and bowl tests. Chimney and cylinder test did not prove useful in our model of mild stroke. In summary, we established the pole test and rotarod as useful tools to evaluate sensory motor deficits early after mild stroke, and corner test and adhesive removal test at later time-points. Alternatively, corner rotation may be a suitable test of long-term function. Test batteries may be further complemented by catwalk and paw preference test for clinically relevant deficits. There was no correlation of behavioral outcome with lesion size at 28 days, and determining whether these tests are useful for detecting a potential benefit of neuroprotective or regenerative therapies requires further testing. [Copyright &y& Elsevier]

Ambivalent effects of interleukin-6 on the pathogenesis of ischaemic stroke have been reported. However, to date, the long-term actions of interleukin-6 after stroke have not been investigated. Here, we subjected interleukin-6 knockout (IL-6−/−) and wild-type control mice to mild brain ischaemia by 30-min filamentous middle cerebral artery occlusion/reperfusion. While ischaemic tissue damage was comparable at early time points, IL-6−/− mice showed significantly increased chronic lesion volumes as well as worse long-term functional outcome. In particular, IL-6−/− mice displayed an impaired angiogenic response to brain ischaemia with reduced numbers of newly generated endothelial cells and decreased density of perfused microvessels along with lower absolute regional cerebral blood flow and reduced vessel responsivity in ischaemic striatum at 4 weeks. Similarly, the early genomic activation of angiogenesis-related gene networks was strongly reduced and the ischaemia-induced signal transducer and activator of transcription 3 activation observed in wild-type mice was almost absent in IL-6−/− mice. In addition, systemic neoangiogenesis was impaired in IL-6−/− mice. Transplantation of interleukin-6 competent bone marrow into IL-6−/− mice (IL-6chi) did not rescue interleukin-6 messenger RNA expression or the early transcriptional activation of angiogenesis after stroke. Accordingly, chronic stroke outcome in IL-6chi mice recapitulated the major effects of interleukin-6 deficiency on post-stroke regeneration with significantly enhanced lesion volumes and reduced vessel densities. Additional in vitro experiments yielded complementary evidence, which showed that after stroke resident brain cells serve as the major source of interleukin-6 in a self-amplifying network. Treatment of primary cortical neurons, mixed glial cultures or immortalized brain endothelia with interleukin 6-induced robust interleukin-6 messenger RNA transcription in each case, whereas oxygen–glucose deprivation did not. However, oxygen–glucose deprivation of organotypic brain slices resulted in strong upregulation of interleukin-6 messenger RNA along with increased transcription of key angiogenesis-associated genes. In conclusion, interleukin-6 produced locally by resident brain cells promotes post-stroke angiogenesis and thereby affords long-term histological and functional protection. [ABSTRACT FROM PUBLISHER]

Abstract: Recent experimental studies in rodents suggest that treatment with inhibitors of phosphodiesterase type 5 (PDE5) (tadalafil, sildenafil, zaprinast) not only increases cerebral blood flow but also improves functional recovery after stroke. Here, we investigated in a mouse model of stroke the effects of vardenafil on survival, functional outcome and lesion size after experimental stroke. Mice were subjected to experimental stroke by occlusion of the middle cerebral artery (MCAO) for 45 min. A group of mice received vardenafil (twice 10 mg/kg body weight per day orally over 14 days) starting 3 h after MCAO. Control animals received the vehicle only. Survival, body weight, and behavior were monitored over 4 weeks and brain lesions were measured by T2-weighted MRI, hematoxylin/eosin — as well as GFAP-staining of cryostat sections, subsequently. The mortality in MCAO-operated animals amounted to 45% until day 10 after stroke and no significant difference in survival between the vardenafil- and vehicle-treatment groups was observed. Compared to sham-operated animals, MCAO-operated mice from both treatment groups demonstrated a significant weight loss until day 5 and regained their body weight by day 14 after ischemia. There was no significant difference between the vardenafil and vehicle-treated MCAO groups. In behavioral studies (sucrose consumption and pole test), analyzing sensorimotor functions as well as a parameter of depression-like symptoms, we observed no significant effect of vardenafil treatment on functional recovery in our model of stroke. Although we observed a trend towards less hemispherical atrophy in the vardenafil compared to the vehicle-treated group four weeks after MCAO our data do not suggest a functionally relevant CNS-tissue protective or regenerative effect in murine stroke. [Copyright &y& Elsevier]

Abstract: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonists (thiazolidinediones) have anti-inflammatory effects and improve endothelium function. Here, we analyzed the effects of pioglitazone on short- and longer-term outcome after mild transient brain ischemia. 129/SV mice were subjected to 30 min filamentous middle cerebral artery occlusion (MCAo), followed by reperfusion. Post event, animals were treated with daily intraperitoneal (i.p.) pioglitazone (20 mg/kg body weight) or vehicle. Pioglitazone given acutely after transient brain ischemia/reperfusion reduced lesion size and the number of Iba1-expressing microglia in the ischemic striatum at three days. In vitro, pioglitazone attenuated migration and proliferation of primary mouse microglia. However, analysis at 6 weeks after MCAo/reperfusion no longer yielded an effect of pioglitazone on either lesion size or Iba1+ cell counts. Regarding functional longer-term outcome, we also did not detect a beneficial effect of pioglitazone on motor function measured either on the pole test or the wire hanging test or on learning and memory in the Morris water maze. Our study thus underscores the importance of extending experimental stroke studies to an analysis of longer-term outcome. [Copyright &y& Elsevier]