Significances were calculated with student test *: models of retinal pathologies, including RP

Significances were calculated with student test *: models of retinal pathologies, including RP. filters in R16 medium as explained 21 (Observe also Physique 2). For the glaucoma model, WT retinas were treated with 50?M NMDA during 48?h, with a medium switch after 24?h. The mouse retinas were cultured for 24?h. Compound 1 was employed at 3.2?M, compound 2 at 10?M and tideglusib at 10?M. Retinas were subsequently fixed SS28 in 4% (wt/vol) paraformaldehyde in phosphate buffer 0.1 M, pH 7.4 for 1?h at RT and processed for the detection of cell death. Open in a separate window Physique 2. Organotypic culture design. (A) The retinas were mounted with the photoreceptors in direct contact to the Teflon disc. (B) After extraction from your eyeball, four cuts were made in the retina to facilitate attachment. Two retinas were cultured in each well. Cell death visualization and Rabbit Polyclonal to CDH23 counting Ganglion cell and photoreceptor cell death was visualized by DNA fragmentation assay terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (DeadEnd Fluorometric TUNEL system; Promega, Madison, WI) as explained 22 . After labeling, the retinas were mounted in Fluoromount-G (Southern Biotechnology, Birmingham, AL), stained with DAPI, and analyzed on a laser confocal microscope (TCS SP5; Leica, Microsystems, Wetzlar, Germany). Image acquisition was performed in four areas of each retina. Serial optical sections were acquired in the depth of the ganglion cell layer or the outer nuclear layer, as decided in studies. The chemical genetic rationale postulates that different small chemical probes assayed in different and/or studies contribute to decipher the role of a potential therapeutic target 23 . Consequently, here we selected three chemically diverse small heterocyclic molecules designed and synthetized in our laboratory that target GSK-3 by different mechanism of inhibition (Physique 1): a substrate competitive inhibitor with an iminothiadiazole scaffold 1 , 24 an ATP competitive inhibitor belonging to the maleimide heterocyclic family 2 , 25 and tideglusib, a non-ATP, non-substrate competitive GSK-3 inhibitor currently in clinical trials for autism spectrum disorders 26 . Tideglusib is a thiadiazolidindione (TDZD) and currently the most advanced compound in clinical development among the selected GSK-3 inhibitors. Additionally, 1, SS28 2 and Tideglusib have previously been tested in cell cultures and animal models showing no toxicity 27C30 . Open in a separate window Figure 1. Chemical structures of the selected candidates and their GSK-3 inhibition features. First we assayed the two more novel inhibitors (1 and 2) in retinal explants obtained from and cultured over Teflon discs (Millipore), as exemplified in Figure 2. The mouse retinal explants are a RP disease model in which there is intrinsic photoreceptor cell death. The retinas were dissected at postnatal day P23, at the peak of cell death 31 , and cultured in the absence or presence of compounds 1 and 2. Cell death was visualized by TUNEL and quantified. Both GSK-3 inhibitors significantly reduced photoreceptor cell death (Figure 3) elicited a neuroprotective action in the RP model. Further, they suggest a novel potential role of GSK-3 inhibition on the treatment of this retinal pathology. Open in a separate window Figure 3. GSK-3 inhibitors decreased photoreceptor cell death in mouse retinal explants. Representative images of groups (A) vehicle, (B) treatment with compound 1 and (C) treatment with compound 2. DCE. Graphic representation of data: (D) mean??standard error is represented for each experimental group. The number in brackets corresponds to the number of retinas; (E) Individual retinal values are depicted. Significances were calculated with student test **: student test *: mouse retinal explants with tideglusib significantly reduced photoreceptor cell death (Figure 5), an observation that reinforces the role of GSK-3 as pharmacological target in retinal RP neuroprotection. Further, it opens an interesting translational opportunity. Tideglusib is an oral drug that has shown a wide safety window in human clinical trials both in Alzheimers disease and SS28 progressive supranuclear palsy 34 , and it is currently on clinical trials for autism spectrum disease 26 . In the light of the results described here, we propose the.