Date of Graduation

Spring 2026

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Paul Durham

Abstract

Migraine and temporomandibular joint disorder (TMD) are debilitating orofacial pain disorders that negatively impact one’s quality of life. These disorders are characterized by elevated levels of adenosine triphosphate (ATP) and activation of central nervous system glial cells that promote and maintain central sensitization and enhance pain signaling in response to inflammatory stimuli. The goal of my study was to characterize the effects of the pro-inflammatory signaling molecule adenosine triphosphate (ATP) on intracellular calcium levels, mRNA levels, and protein expression in astrocytes, microglia, and oligodendrocytes to better understand the role of ATP in promoting central sensitization. To address this goal, primary cultures of brainstem tissue were established from male and female Sprague Dawley neonatal rats. Changes in intracellular calcium levels in glial cells were investigated using the ratiometric dye Fura-2 and fluorescent microscopy, while mRNA levels were determined by RT-qPCR and protein expression by immunocytochemistry. Following initial plating, astrocytes and microglia, which comprised most of the glial cells, continued to proliferate until reaching near confluency by day 8 post plating. On day 8, all three glial cell types were found to express P2X3 and P2X7 ATP-gated calcium channel receptors on their cell body and processes. Incubation of cultures with 100 µM ATP increased the amplitude and duration of the intracellular calcium response in astrocytes and microglia. Although under the culture conditions all three glial cells types were present on day 8, the percentage of astrocytes was much greater than the other glial cell types. In addition, mRNA and protein expression of key proteins implicated in central sensitization were greatly suppressed in day 8 cultured cells when compared to in vivo brainstem tissues, likely due to high levels of inducible nitric oxide synthase expression. Findings from my study demonstrated ATP stimulation of intracellular calcium levels in astrocytes and microglia, which would be associated with glial activation, is a key event in the development and maintenance of central sensitization in migraine and TMD.

Keywords

Adenosine triphosphate, calcium, central sensitization, migraine, temporomandibular joint disorder, pain, astrocytes, microglia

Subject Categories

Cell Biology

Copyright

© Donovan J. Aardema Faigh

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