Undergraduate Honors Theses

Thesis Defended

Spring 2011

Document Type

Thesis

First Advisor

false

Abstract

Endothelial cells are especially important within the central nervous system as they line capillaries and form the blood-brain barrier. Moreover, they are the first cell type within the CNS to be exposed to foreign pathogens and drugs in the bloodstream. In other cell types in the CNS, activation of Toll-like Receptor 4 (TLR4) results in activation of transcription factors such as NF-κB and AP-1, both known to induce the production of pro-inflammatory cytokines. The expression of pro-inflammatory cytokines sensitizes neurons, thus decreasing pain thresholds and prolonging pain. Interestingly, recent research demonstrates that opioids (analgesics prescribed for chronic pain) not only bind the µ-opioid receptor to confer pain relief, but also activate TLR4. It is thought that TLR4 activation, and subsequent release of pro-inflammatory cytokines, contributes to the negative effects of tolerance, dependence, and withdrawal associated with morphine use. Because of the dearth of research surrounding opioids and their effect on this cell type, this study attempted to characterize TLR4 activation in endothelial cells. Endothelial cells were isolated from the brain and spinal cord and incubated with various doses of (+)-morphine. Results indicate that TLR4 is indeed activated by (+)-morphine, producing PGE2 and increasing the mRNA of IκB, CD14, and TLR4 at 100 µM (+) morphine 24 h after incubation. The pro-inflammatory response induced by 100 µM (+)-morphine can be blocked with both PI3K and IκB-α inhibitors, significantly reducing NF-κB activation and PGE2 production. Furthermore, signal transduction occurs in a MyD88-dependent fashion as CLI-095, a TLR4 signaling inhibitor that blocks the MyD88-independent pathway, had no significant effect on mRNA expression or PGE2 production. These data suggest that (+)-morphine induces TLR4 activation in endothelial cells and, in concert with other cell types in the central nervous system, may contribute to the negative effects of morphine use.

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