Thursday, April 17, 2014

HIV-1R Viral Protein R and Memory Impairment

Using Synpatophysin as Marker for Synaptic Loss

Our Neuron-Glial Markers continue to shine in challenging applications. Here researchers examined whether infusion of the Vpr-expressing astrocytes affected synaptophysin expression in the hippocampus. The authors of the study, using Neuromics' Mouse Monoclonal Synaptophysin Antibody,  found a significant reduction in synaptophysin staining in CA3: Lilith Torres and Richard J Noel. Astrocytic expression of HIV-1 viral protein R in the hippocampus causes chromatolysis, synaptic loss and memory impairment. Journal of Neuroinflammation 2014, 11:53 doi:10.1186/1742-2094-11-53.

Images: Astrocytic HIV-1 viral protein R (Vpr) expression decreased synaptophysin immunoreactivity (A) Representative light photomicrograph showing the distribution of synaptophysin immunoreactivity in the rat hippocampal CA3 formation. Green fluorescent protein (GFP) right side. Vpr shows both left and right. Magnification 100×. (B) Densitometric analysis revealed significantly decreased mean value for the Vpr group compare to control.

Protocol: To examine changes in synaptophysin between control and HIV-1 Vpr exposed rats, tissue sections from each group were processed for immunocytochemistry. The samples were cut at 4 μm thickness with a microtome (Microm HM340, Microm International) and fixed to positively charged microscope slides. Fixed tissues were deparaffinized in xylene substitute for 30 minutes, rehydrated through graded alcohols and neutralized with 3% hydrogen peroxide (Sigma-Aldrich), followed by a rinse under running tap water and immersion in antigenretrieval solution (0.01 M citrate, pH 6.0) for 1 minute at 98°C. Then sections were washed in TBS for 5 minutes and treated with blocking solution containing normal goat serum (BioGenex, cat# HK112-9KE). Sections were incubated for 24 hrs at 4°C in mouse monoclonal antisynaptophysin antibody (Neuromics, cat # MO20000, 1:500 dilutions). Negative controls with TBS instead of primary antibody were run in each slide. Primary antibody was washed in TBS buffer for 2 × 5 minutes and incubated with Multi Link secondary antibody (Super Sensitive Link-Label IHC Detection System, cat# LP000- ULE, BioGenex, San Ramon, CA, USA). Secondary antibody was washed in TBS and incubated in ABC-HRP, washed in TBS buffer and incubated in 3,3′-diaminobenzidine (cat# HK153-5KE, Biogenex, San Ramon, CA, USA). Slides were rinsed in water and counterstained with hematoxylin for 30 sec. The sections were rinsed, dehydrated and and mounted with Cytoseal XYL (cat# 8312-4, Richard Allan Scientific, Kalamazoo, MI, USA). For quantitative densitometry, images of regions of interest (ROI) in the CA3 were captured from 5 rats in each group using NIH Image J 1.50 software.

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