Technical Reference #1909

1909.

Evidence that opioids may have toll-like receptor 4 and MD-2 effects M.R. Hutchinson; Y. Zhang; M. Shridhar; J.H. Evans; M.M. Buchanan; T.X. Zhao; P.F. Slivka; B.D. Coats; N. Rezvani; J. Wieseler; T.S. Hughes; K.E. Landgraf; S. Chan; S. Fong; S. Phipps; J.J. Falke; L.A. Leinwand; S.F. Maier; H. Yin; K.C. Rice; L.R. Watkins, University of Colorado at Boulder, Brain; Behavior; and Immunity, 24(1909), (2010)
Link To Paper

Keywords:
Toll-like receptor 4; Knockout; Opioid; Analgesia; Dependence; Glia; Microglia

Materials & Methods:
TLR4 signaling leads to the simultaneous activation of three parallel intra-cellular signaling pathways. Two of these (through NF-jB and MAPK) are principally responsible for the proinflammatory responses induced by TLR4 activation while the third parallel pathway (PI3K/Akt1) is more related to cell survival apoptosis and cell motility (Dauphinee and Karsan 2006; Laird et al. 2009). As all three are activated by agonism at TLR4 any can be used as a reflection of TLR4 activation. Given the availability of a RAW264.7 mouse macrophage cell line stably transfected to express green fluorescent protein (GFP)-tagged Akt1 (Evans and Falke 2007) mobilization and cytosolic clearance of GFP-Akt1 was used as an indicator of TLR4 activation. Cells were grown up in growth media and then were plated at 2 105 cells/mL density in growth media on 35 mm MatTek Glass Bottom Dishes (Ashland MA USA) for 18 h prior to imaging. Just prior to imaging the growth mediawas removed from the plates washed two times with HBSS supplemented with 25 mM HEPES buffered to pH 7.4 and replaced with 1 mL warmed conditioned imaging Hanks Buffer media (media was conditioned by a 24 h incubation with RAW264.7 cells). Imaging was carried out on a Nikon inverted microscope (Melville NY USA) with a 60 oil immersion objective GFP/RFP dichroic mirror with corresponding single band excitation and emission filters (Chroma Technology) and CoolSNAP ES camera (Photometrics Tucson AZ USA). A mercury lamp provided excitation. Images were captured every 7.5 s. Baseline fluorescence was captured for five frames following which vehicle or antagonist was added in 200 ll. Imaging continued for a further 20 frames at which time LPS or test agonist (200 ll) were applied and monitored for a further 20 frames. If no visual response was obtained C5a (200 ll) was added to the plates to confirm if the cells were responsive. GFP-Akt1 cytosolic clearance was quantified using ImageJ and expressed as a normalized change in cytoplasmic fluorescence over time.

Microscopic Technique
Inverted Microscopy

Cell Type(s)
RAW264.7 mouse macrophage cell line