Pharmacological studies using bradykinin B 2 receptor antagonists suggest that bradykinin, an early mediator of inflammation and the main metabolite of the kallikrein-kinin system, is involved in secondary brain damage after cerebral ischemia. However, the time-course of bradykinin production and kinin receptor expression as well as the conclusive role of bradykinin B 2 receptors for brain damage after experimental stroke have not been elucidated so far. C57/Bl6 mice were subjected to 45 mins of middle cerebral artery occlusion (MCAO) and 2, 4, 8, 24, and 48 h later brains were removed for the analysis of tissue bradykinin concentration and kinin B 2 receptor mRNA and protein expression. Brain edema, infarct volume, functional outcome, and long-term survival were assessed in WT and B 2 À/À mice 24 h or 7 days after MCAO. Tissue bradykinin was maximally increased 12 h after ischemia (three-fold), while kinin B 2 receptor mRNA upregulation peaked 24 to 48 h after MCAO (10-to 12-fold versus naïve brain tissue). Immunohistochemistry revealed that kinin B 2 receptors were constitutively and widely expressed in mouse brain, were upregulated 2 h after ischemia in cells showing signs of ischemic damage, and remained upregulated in the penumbra up to 24 h after ischemia. B 2 À/À mice had improved motor function (Po0.05), smaller infarct volumes (À38%; Po0.01), developed less brain edema (À87%; Po0.05), and survived longer (Po0.01) as compared with wild-type controls. The current results show that bradykinin is produced in the brain, kinin B 2 receptors are upregulated on dying cells, and B 2 receptors are involved in cell death and brain edema formation after experimental stroke.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 1015 photons.cm−2.s−1. Vector doses of 5 × 1010 and 5 × 1011 vg/eye transfected up to 7000 RGCs/mm2 in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.

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Aims/hypothesis: The aim of this study was to investigate the effects of liver X receptor (LXR) activation on lipid metabolism and insulin action in human skeletal muscle cells prepared from control subjects and from patients with type 2 diabetes. Subjects and methods: Cultured myotubes were obtained from muscle biopsies of 11 lean, healthy control subjects and ten patients with type 2 diabetes. The mRNA levels of LXR isoforms and lipogenic genes were estimated by RT-quantitative PCR, and the effects of LXR agonists on insulin action were evaluated by assays of protein kinase B serine 473 phosphorylation and glycogen synthesis. Results: Both LXRα and LXRβ were expressed in human skeletal muscle and adipose tissue and there was no difference in their mRNA abundance in tissues from patients with type 2 diabetes compared with control subjects. In cultured muscle cells, LXR activation by T0901317 strongly increased expression of the genes encoding lipogenic enzymes, including sterol regulatory element binding protein 1c, fatty acid synthase and stearoyl-CoA desaturase 1, and also promoted triglyceride accumulation in the presence of a high glucose concentration. Importantly, these effects on lipid metabolism did not affect protein kinase B activation by insulin. Furthermore, LXR agonists did not modify insulin action in muscle cells from patients with type 2 diabetes. Conclusions/interpretation: These data suggest that LXR agonists may lead to increased utilisation of lipids and glucose in muscle cells without affecting the mechanism of action of insulin. However, the long-term consequences of triglyceride accumulation in muscle should be evaluated before the development of effective LXR-based therapeutic agents.