The present study was designed primarily to assess associations with ABCB1 3435CT

et of experiments Neuroprotective Effects of TrkB Agonist 29D7 Caspase-3 Activity Assay Twenty-four hours after H-I, the hippocampal and cortical tissues from both lesioned and unlesioned hemispheres were Halofuginone cost rapidly dissected and Asp-Glu-Val-Asp- cleavage activities were measured as previously reported. Tissue lysates were incubated Neuroprotective Effects of TrkB Agonist 29D7 with 90 ml of an assay buffer containing 30 mM acetyl-DEVD-AMC and the enzymatic activities were calculated by kinetic analyses of the emitted fluorescence measured every 5 min for 30 min. Acetyl-AMC was used to obtain a standard curve and the enzyme activity was presented as picomoles of AMC per milligram of protein per minute. variance followed by Dunnett’s multiple comparison method. P value,0.05 was considered significant. Results 29D7 Activates ERK1/2 and AKT in Neurons of the Neonatal Brain Previous studies have demonstrated that endogenous TrkB agonists such as BDNF can activate both the ERK1/2 and PI3K-AKT pathways in responsive cells. We first asked whether the TrkB agonist antibody 29D7 could activate these pathways in the neonatal brain in vivo. Western blot analyses revealed that icv injection of 29D7 increased ERK1/ 2 phosphorylation and AKT phosphorylation in the cortex ipsilateral to the icv injection. Levels of both phosphorylated ERK1 and phosphorylated ERK2 were significantly increased by 1 h and lasted up to 24 h after the injection. Similarly, 29D7 significantly PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19638978 increased ATK phosphorylation at least for up to 24 h. We also found that increased phosphorylation of ERK1/2 and AKT was noted in the hemisphere contralateral to the injection, indicating that the antibody could reach to the entire brain after icv injection. However, icv injection of control IgG was no effect on the levels of phosphorylated ERK1/2 and phosphorylated AKT for up to 24 h. Based on the fact that TrkB is expressed in neurons in the brain, we hypothesized that activation of ERK1/2 and AKT by 29D7 may primarily occur in neurons. To address this, brain tissues were prepared at various timepoints after icv injection of 29D7 and subjected to immunohistochemical analyses. Similarly to our previous report, phosphorylated ERK1/2 -immunoreactivity was detectable mostly in neuronal cell processes in the IgG-treated P7 control rats with no obvious staining in the cell bodies or nuclei. However, upon icv injection of 29D7, pERK1/2-IR was greatly increased in all cellular compartments, including neuronal PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639654 processes, cell bodies, and nuclei in neuronal layers of the cortex and the hippocampus. The prolonged increase in the pERK1/2-IR was noted in neuronal cell processes through all layers in the cortex 24 h after the injection. Double labeling with a neuron-specific marker, NeuN, revealed that pERK1/2-IR was localized to neurons. Assessment of Brain Damage following Hypoxic-ischemic Injury Regional brain loss was determined at 7d or 35d post H-I injury by calculating the amount of surviving tissue in coronal sections as previously described. Briefly, coronal sections from the genu of the corpus callosum to the end of the dorsal hippocampus were stained with cresyl violet. The cross-sectional areas of the striatum, cortex and hippocampus in each of eight equally spaced reference planes were photo-scanned and the area of each brain region was calculated using Adobe Photoshop 6.0. The sections utilized for quantification in a blinded manner corresponded approximately to Plates 20, 25, 30, 35, 5