Poster Presentations: P1 pathways. Since brain inflammation and hippocampal neurogenesis may correlate with bioenergetic function, we also analyzed genes and proteins that reflect neurogenesis and brain inflammation activity. Results: 21month old C57BL/6 mice showed increased brain PGC-1a protein, mTOR and phospho-mTOR protein, citrate synthase mRNA, and mtDNA copy number. Hippocampal VEGF-A gene expression trended higher, and a positive correlation between VEGF-A and PRC mRNA levels was observed. Brain DCX, BDNF, TNF-a, and CCL11 gene expression, as well as plasma CCL11 protein levels, were unchanged. Despite these apparent negative findings, a negative correlation between plasma CCL11 protein levels and hippocampal DCX gene expression was observed; further analysis indicated exercise may mitigate this relationship. Conclusions: Supra-lactate threshold exercise beneficially impacts bioenergetic-relevant parameters in the brains of aged mice. Our data further suggest supra-lactate threshold exercise may enhance neurogenesis by uncoupling it from the effects of inflammatory chemokines. This study supports the view that exercising above the lactate threshold may benefit the aging brain.
P1-418
SUSTAINED DELIVERY OF SURFACE MODIFIED DONEPEZIL HCL LOADED PCL NANOPARTICLES FOR ALZHEIMER’S DISEASE
Muthukumar Amirthalingam1, Udupa Nayanabhirama2, Srinivas Mutalik1, 1Manipal College of Pharmaceutical Sciences, Manipal, India; 2Manipal University, Manipal, India. Contact e-mail: muthukumar.
[email protected] Background: Donepezil HCl is an AChE inhibitor used in the treatment of mild to moderate Alzheimer’s disease. The objective of the study was to prepare receptor targeted sustained release nanoparticles for donepezil HCl using a biodegradable polymer. The surface modification targets the nanoparticles through receptor-mediated endocytosis across the BBB and releases the drug in a sustained manner with low dose compared to the available oral dosage form. Methods: PCL nanoparticles were prepared by nanoprecipitation technique using design of experiments. The effect of drug-polymer ratio, homogenization speed and time were studied. The encapsulation efficiency was analysed by HPLC. Particle size and zeta potential were analysed by DLS method. The morphology of the nanoparticles was studied by SEM. The prepared nanoparticles were surface modified with Polysorbate-80 and in-vitro dissolution study was carried out. The acetylcholinesterase inhibition assay was carried out using Electrophorus electricus AChE enzyme. The invitro cell line toxicity was conducted in Vero cells. Nanotoxicity analysis was studied on zebra fish eggs and compared with untreated control. Results: The formulated nanoparticles were in the size between 363 nm to 501 nm. The SEM analysis showed that the particles were spherical in shape and surface was smooth. The in-vitro drug release of the nanoparticles was sustained and the drug was stable in the formulation throughout the study period. The PCL nanoparticles showed an AChE inhibitory effect on the selected enzyme. The nanoparticles were found to be safe on the Vero cells. The nanotoxicity study of the PCL nanoparticles on the zebra fish eggs was found to be safe for the larvae. The larvae development, movement and hatching from the eggs were found to be comparable with the control. Conclusions: The prepared PCL nanoparticles were able to release the drug in a sustained manner. The surface modification will target the nanoparticles to the brain for effective AChE inhibitory activity. The PCL nanoparticles were found to be safe on the tested in-vitro cell line and zebra fish larvae.
P1-419
ALPHA-7 NICOTINIC RECEPTOR STIMULATION OFFERS NEUROPROTECTION IN A MODEL OF ALZHEIMER’S DISEASE BASED ON OXIDATIVE STRESS
P467
Elisa Navarro, Izaskun Buendia, Esther Parada, Javier Egea, Manuela G. Lopez, Instituto Teofilo Hernando, Universidad Autonoma de Madrid, Madrid, Spain. Contact e-mail:
[email protected] Background: There is increasing evidence about the role of oxidative stress in neurodegeneration and Alzheimer’s disease (AD). However, the majority of studies and models of AD focus on amyloid beta and tau pathology. Methods: For this purpose, rat organotypic hippocampal cultures (OHCs) were exposed during 4 days to subtoxic concentrations of lipopolysaccharide and antimycin A (LPS/AA). Cell death and reactive oxygen species (ROS) production were quantified using propidium iodide and DCFDA fluorescence staining. Hemoxigenase-1 (HO-1) expression and hyperphosphorylated tau were measured by western blot. Protein aggregates were studied using Thioflavin-S inmunofluorescence. Results: We obtained that neither LPS (1 ng/ml) nor AA (0.1 uM) alone, caused neurotoxicity or increase of oxidative stress markers. However, the combination of these two stimuli at subtoxic concentrations caused a synergistic neurotoxic effect, in addition to mitochondrial depolarisation and overproduction of ROS, which revealed mitochondrial dysfunction. Most interesting were the findings that exposure of OHCs to these subtoxic concentrations of LPS/AA were sufficient to trigger pathological features associated to AD such as aberrant protein aggregates and Tau hyperphosphorylation. In this AD-like in vitro model, PNU282987 (an a7 nicotinic receptor agonist) prevented the neurotoxicity and the pathological hallmarks elicited by the combination of LPS/AA. The latter effects were a7 nAChR specific and dependent of the expression of HO-1 as the protection and ROS production were avoided using abungarotoxin (a7 nAChR antagonist) and SnPP (HO-1 inhibitor). Conclusions: Taken together, these results demonstrate that oxidative stress is a key element in the pathological cascade of AD and, cholinergic strategies based on a7 nAChRs activation can ameliorate these alterations by a mechanism that implicates hemoxigenase-1 induction. Moreover, we have developed a suitable in vitro AD model useful for drug screening. P1-420
COMBINATION OF SUBEFFECTIVE CONCENTRATIONS OF MELATONIN AND GALANTAMINE IMPROVES ALZHEIMER’S LIKE PATHOLOGY IN ORGANOTYPIC HIPPOCAMPAL SLICES EXPOSED TO THE ASSOCIATION OF B-AMYLOID AND OKADAIC ACID
Izaskun Buendia, Elisa Navarro, Javier Egea, Esther Parada, Manuela G. Lopez, Instituto Teofilo Hernando-Universidad Autonoma de Madrid, Madrid, Spain. Contact e-mail:
[email protected] Background: Although many compounds have been evaluated in AD, there is still no effective treatment. One of the main problems to evaluate new drugs is the lack of good experimental in vitro models. Melatonin is a neurohormone whose levels are significantly reduced or absent in AD patients. Acetylcholinesterase (ACE) inhibitors cause overexpression of ACE, and because of their side effects, therapeutic compliance is limited; these effects could account, in part, for their limited efficacy in clinic. Methods: Cell death was measured as the increase in propidium iodide uptake, thioflavin S staining was used to detect Ab aggregates, western blot to detect hyperphosphorylated Tau protein and iNOS expression, fluorescent dyes to determine oxidative stress and NO production, Q-PCR to measure IL-1b expression and Iba-1 and GFPA immunofluorescent staining to observe morphological changes in microglia and astrocytes, respectively. Results: The association of subtoxic concentrations (0’5mM Ab /1nM O.A) for four days in OHC causes a synergic cell death, which is mainly apoptotic. In addition, thioflavin S aggregates, hyperphosphorylation of Tau protein, oxidative stress, neuroinflammation, microgliosis and astrogliosis were shown. Therefore, this in vitro model reproduced many of the pathological markers observed in AD and, could be used as a model to test new compounds or new pharmacological strategies before moving into in vivo models. In addition, our results show that
