The 50% paw withdrawal threshold in response to a series of eight von Frey hairs (0.41 to 15.10 g.) was examined by the up-down method as explained previously18. well as reduced TNF expression in DRG. Intrathecal injection of MCP-1 neutralizing antibodies reduced paclitaxel-induced macrophage recruitment into the DRG and also blocked the behavioral indicators of CIPN. Intrathecal treatment with the TLR4 antagonist LPS-RS blocked mechanical hypersensitivity, Alprenolol hydrochloride reduced MCP-1 expression, and blocked the infiltration of macrophages into the DRG in paclitaxel treated rats. Finally, the inhibition of macrophage infiltration into DRG following paclitaxel treatment with clodronate or LPS-RS prevented the loss of intra-epidermal nerve fibers (IENFs) observed following paclitaxel treatment alone. Taken altogether, these results are the first to show a mechanistic link such that activation of TLR4 by paclitaxel prospects to increased expression of MCP-1 by DRG neurons resulting in macrophage infiltration to the DRG that express inflammatory cytokines and the combination of these events results in IENF loss and the development of behavioral indicators of CIPN. (LPS-RS, 20 g in 20 L PBS; InvivoGen, San Diego, CA) or anti-MCP-1 neutralized antibody (200 g/mL, 20 L per application; AbD Serote, Raleigh, NC) or equivalent amount of nonspecific IgG (Rabbit IgG, Jackson ImmunoResearch, West Grove, PA) were delivered intrathecally 24 hours prior to the first injection of paclitaxel and was continued Alprenolol hydrochloride once daily for the next 7 days for a total of 8 injections (day 0C7). Intrathecal injection was given 30 minutes prior to paclitaxel when both drugs were administered on the same day. 2.4 Intravenous injection of clodronate The macrophage toxin clodronate in liposomes (clophosome-A, 7mg/ml clodronate disodium) or control liposome (FormuMax, Sunnyvale, CA) were intravenously administrated to 24 rats (12 verses 12) with the volume of 0.8 ml on day 7 and day 10 in paclitaxel treated rats. 2.5 Mechanical withdrawal threshold Mechanical withdrawal threshold was tested before, during and following paclitaxel treatment by an experimenter blinded to treatment groups. The 50% paw withdrawal threshold in response to a series of eight von Frey hairs (0.41 to 15.10 g.) was examined by the up-down method as explained previously18. Animals were placed under obvious acrylic cages atop a wire mesh floor. Beginning with a filament with a bending pressure of 2.0 g., the filaments were applied to the paw just below the pads with no acceleration at a pressure just sufficient to produce a bend and held for 6C8 sec. A quick flick or full withdrawal was considered a response, in which case the next lower filament was applied. If no response was observed then the GCSF next higher filament was applied. This continued until three responses to a single filament were observed. The test will be performed three hours after drugs application on those days when both happened. 2.6 Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital (Nembutal, 50 mg/kg, i.p., Lundbeck, Inc., Deerfield, IL) at days 3, 7, 14 and 21 Alprenolol hydrochloride after paclitaxel treatment. Then they were perfused through the ascending aorta with warm saline followed by chilly 4% paraformaldehyde in 0.1 M PBS. The L4 and L5 DRG, spinal cord, the spleen and the hindpaw foot pad were removed, fixed in 4% paraformaldehyde for 6 hours, and then cryo-protected in 30% sucrose answer. Tissue blocks were then submerged in optimal cutting heat (OTC) medium and frozen. Transverse spleen and spinal cord sections (20 m), longitudinal DRG sections (8 m), and hindpaw epidermal cross-sections (14 Alprenolol hydrochloride m) were cut in a cryostat, mounted on gelatin-coated glass slides (Southern Biotech, Birmingham, AL), Alprenolol hydrochloride and processed for immunofluorescent labeling. After blocking in 5% normal donkey serum and 0.2% Triton X-100 in PBS for one hour at room temperatures, DRG sections had been incubated overnight at 4C in 1% normal donkey serum and 0.2% Triton X-100 in PBS containing major antibodies against Compact disc68 to visualize macrophages (mouse, 1:500; Abcam, Cambridge, MA), MCP-1 (rabbit, 1:500; AbD Serotec, Raleigh, NC) or anti-TNF (goat, 1:200; Santa Cruz, Dallas, TX). The.