13778; Thermo Fisher Scientific) diluted in Opti-MEM I reduced Serum medium (cat. was further explored by examining its receptors and their function. We found that netrin-1 infusion improved neurological function, attenuated secondary impairment of BBB by up-regulating the levels of tight junction proteins and diminishing extravasation of albumin, with autophagy activation 14 days after MCAO. Netrin-1 also enhanced cell survival and autophagy activity in OGD-treated cells, inhibited by UNC5H2 siRNA transfection. Furthermore, the beneficial effects of netrin-1 were suppressed by PI3K inhibitors 3-Methyladenine and LY294002. Our results showed that netrin-1 ameliorated BBB impairment secondary to ischemic stroke by promoting tight junction function and endothelial survival. PI3K-mediated autophagy activation depending on UNC5H2 receptor could be an underlying mechanism. and = 24), 50 g/mL netrin-1 plus 200 nmoL PI3K inhibitor 3-Methyladenine (3-MA) (cat. No. M9281; Sigma-Aldrich; = 24) or phosphate-buffered saline as vehicle (= 24) 24 h later. The infusions (12 l/d over a period of 7 days) were made using a 1007D Alzet osmotic minipump (Durect, Cupertino, CA, USA) at the following stereotaxic coordinates: 0.8 mm posterior to the bregma, 1.4 mm lateral to the bregma on the right side and 3.6 mm below the dura. The bioactivity of reagents in the present regimen has been verified by our previous study (Liao et al., 2013). Neurological evaluation Neurological function was evaluated blindly in each group before and 1, 8, and 14 days after MCAO with a altered neurologic severity score (mNSS), which included a combination of motor and sensory functions, balance, and reflex assessments (Chen et al., 2014). The mNSS was recorded from 0 (normal) to 18 (maximal deficit), with 13C18 as severe injury, 7C12 as moderate injury, and 1C6 as moderate injury. Tissue preparation Eight and 14 days after actual or sham MCAO, 12 rats from each group were anesthetized and sacrificed. For H&E staining and immunofluorescence, the rat brains from six rats were transcardially perfused and post-fixed with 4% paraformaldehyde at 4C for 8 h. Series of adjacent 10 m coronal frozen sections were collected at the ipsilateral thalamus level. For western blot, the rat brains from your other six rats were transcardially perfused with heparinized saline at 4C. The ipsilateral thalamus was quickly obtained and frozen in liquid nitrogen, and then stored at ?80C. To observe the microstructure of tight junctions, small blocks from your ipsilateral thalamus (= 3) were fixed, dehydrated, and embedded for transmission electron microscopy. Series of adjacent 80 nm sections were made using Tonabersat (SB-220453) an Ultracut-E ultramicrotome (Reichert-Jung, Vienna, Austria), and viewed Tonabersat (SB-220453) under a LM-10 electron microscope (Philips, Amsterdam, Holland) at 1,700 magnification. Tight junctions appear as a series of discrete sites of apparent membrane fusion (kissing points) between the outer leaflets of the plasma membranes of adjacent cells. Cell culture and oxygen-glucose deprivation (OGD) Rat brain microvascular endothelial cells (RBMVECs) (cat. No. R840-05a; Cell Application) were grown and managed in Dulbecco’s altered eagle medium (cat. No. 11885-084; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in a humidified incubator under 5% CO2 at 37C. Cells were split at 70C80% confluence before the following experiments. OGD is Tonabersat (SB-220453) used to mimic ischemic conditions as previously explained (Park et al., 2005). In brief, RBMVECs were gently washed twice with glucose-free Dulbecco’s altered eagle medium (cat. No. 11966-025; Thermo Fisher Scientific), and placed in a modular chamber with Rabbit Polyclonal to CSTF2T dual circulation meter (Billups-Rothenberg, Del Mar, CA, USA). Cells in the chamber were flushed with 95%N2/5% CO2 gas combination at a circulation rate of 4L/min for 10 min to produce Tonabersat (SB-220453) hypoxic conditions, and then incubated at 37C for 1 h. Hypoxic conditions within the chamber were monitored using a gas analyzer (Coy Laboratory, Grass Lake, MI, USA). The extent of OGD-induced death of cells was dependent on the duration of OGD, and OGD for 1 h is at a critical threshold to induce pivotal signaling events for cells in the current method. Control cells were treated without OGD condition. To elucidate the role of netrin-1 on RBMVECs and possible involvement of PI3K pathway, cells were pre-treated with 50 ng/mL netrin-1 (R&D System), 20 mol/L PI3K inhibitor LY294002 (cat. No. L9908; Sigma-Aldrich), netrin-1 plus LY294002, or only equivalent amount of diluent answer for 2 h before OGD. The used concentrations of reagents were based on previous studies and were effective for its physiological function (Park et al., 2004; Wilson et al., 2006)..