Neuroinflammation is a common pathophysiological feature of many disorders affecting the central nervous system, including migraine—one of the most prevalent neurological conditions, which significantly impairs quality of life, particularly when it progresses to the chronic form. The aim of the present study was to analyze oxidative changes following a single administration of nitroglycerin (NTG), as well as to investigate alterations in the glial microenvironment and inflammatory processes induced by chronic NTG administration. Registration of biosensor signals (HyPer7 and SypHer3s) in vivo did not reveal changes in hydrogen peroxide levels or pH following single NTG administration in striatum and cortex. In contrast, analysis of chronic NTG administration indicates neuroinflammatory processes occurring in the thalamus and the dentate gyrus of the hippocampus, but not in the somatosensory cortex without disruption of the BBB and decreased degranulation of meningeal mast cells. We observed a decrease in the mRNA expression in the thalamic tissue of the neuroprotective transforming growth factor beta 1 gene and an increase in the expression of the pro-inflammatory interferon gamma. The regional specificity of neuroinflammation supports the suggestion that maladaptive changes in these structures could play a critical role in the transition from episodic to chronic migraine.