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Publication Date

2008-06-25

Availability

UM campus only

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

2008-05-05

First Committee Member

Glenn W. Kerrick - Committee Chair

Second Committee Member

Irene Litosch - Committee Member

Third Committee Member

Robert Keane - Committee Member

Fourth Committee Member

Kenneth J. Muller - Mentor

Fifth Committee Member

Wenbiao Gan - Outside Committee Member

Abstract

In both vertebrates and invertebrates, including leeches, microglia are rapidly activated by central nervous system (CNS) damage and migrate to the lesions. Adenosine triphosphate (ATP), nitric oxide (NO) and endocannabinoids have been implicated in controlling activation and migration, but details of the mechanisms are uncertain. This dissertation tests the hypothesis that endocannabinoids coordinate and influence the microglial response to nerve cord crushing. Chapter 1 reports that application of endocannabinoids to nerve cords at concentrations as low as 100 nM for arachidonylethanolamide (anandamide, or AEA) reduced the number of migrating microglia, but not when cords were pretreated with 10 µM of the CB2 cannabinoid receptor (CB2R) antagonist SR144528. In addition, immunoblots confirmed the expression of CB1-like and CB2-like receptors and immunohistochemistry showed that they were concentrated at lesions, where microglia accumulated. Benzoyl ATP (BzATP) also reduced microglia accumulation, an effect blocked by pretreatment of nerve cords with SR144528, whereas the G-protein coupled P2YR agonists uridine triphosphate (UTP) and methylthio-ATP (MeSATP) at 100 µM did not reduce accumulation. This result suggested that P2X7R activation elicited production and release of a CB2R agonist that influenced microglia movement. Chapter 2 reports that extracellular ATP levels were highest in the CNS within the first 30 min of injury and remained above unharmed controls for at least 2 hours. Application of 10 units (U) of the ATPase apyrase to nerve cords reduced accumulation of microglia at lesions, another indication that microglia require extracellular ATP to accumulate. Chapter 3 reports that AEA stopped ATP-induced movement of microglia, and that this effect was blocked by pretreatment of nerve cords with the CB1R antagonist SR141716A (10µM), the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L NAME, 2 mM) or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide (carboxy-PTIO or cPTIO, 1 mM). Thus the migration of microglia to lesions is regulated not only by ATP acting on P2YR and by NO acting on soluble guanylate cyclase (sGC), but also potentially by ATP binding to P2X7-like receptors to increase the production of cannabinoids. Cannabinoids, binding to the CB1R and CB2R cause production of NO, which suppresses microglia movement.

Keywords

Central Nervous System; Leech; Anandamide; Cannabinoids; Migration; Microglia

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