Treatment with Nicotine derived Nitrosamine Ketone NNK Causes Disruption of Blood Brain Barrier BBB and Microglia Activation in Mice

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a nicotine metabolite produced within the tobacco plant, from combustion, and from metabolic breakdown. Cigarette Smoke (CS) continues to be a leading cause for decline of quality of life as well as deaths globally. While the link to poor health and eventually early death has been accepted for decades, it is increasingly recognized that smoking may contribute to a broad range of disorders. Epidemiologically, CS has been associated with neuroinflammation and several neurological disorders including Alzheimer’s disease, stroke, and multiple sclerosis. While direct links are not fully understood, studies in a humanized flow-based in vitro blood-brain barrier model used CS extract to show that CS can have pro-inflammatory effects and promote loss of BBB function and viability. Moreover, other studies have found that CS extract exacerbates hyperpermeability of cerebral endothelial pointing to disruption of the integrity of the BBB. CS is also linked to increased oxidative stress in neurons, microglia, and astrocytes in both in vivo and preclinical models. In one study, NNK was suggested to induce glial activation sustained from 4 to 12 days when given IP in mice. While studies of the direct effects of CS are valuable the composition of chemicals is complex and highly variable. Another approach is to study the effect of a prominent component. Extensive cancer literature has established NNK as an important component of CS for having a direct effect on DNA mutation and its ability to bind and activate nicotinic acetylcholine receptors (nAChRs). However, little is known about the specific effects of NNK on the BBB or microglial dynamics particularly as they relate to the vasculature and across multiple areas. Here we show that NNK when given through an intranasal route, both in acute (4 days) and chronic (12 weeks) exposures, leads to both disruption of the BBB and vessel-localized microglia activation that is highly localized to the vasculature, the latter of which shows increased vasospastic activity. To investigate microglial and vascular responses to intranasal NNK in vivo, we combined the use of a transgenic mouse line (CX3CR1-GFP) with intravital neuroimaging. Single volumetric images and time series of up to an hour were taken to assess spatial and dynamic observations. A machine learning algorithm was employed to segment microglial soma from processes and analyses were performed to evaluate morphometry as well as associations with the vasculature. Results indicate that microglia activation was found to be heterogeneous; that is activated microglia and ramified microglia existed within the same field of view with activated glia in contact or localized to the vasculature. NNK-treated animals displayed a quantitative increase in vessel-associated microglia (VAM) compared to PBS controls. Temporal analysis in those sites showed VAM microglial sustained an ameboid appearance over time and clustered near the vessels. An increase in vascular events, including vasoconstriction, vasodilation, and microbursts, were observed in NNK-treated animals suggesting a pronounced effect of NNK in vasculature. In chronic treatment, the mentioned effects were sustained and in some cases exacerbated. Taken together, these results suggest an immunomodulatory effect of NNK marked by sporadic and highly localized microglia activity accompanied by vascular events which over the long term can produce pathophysiology that could contribute to or exacerbate ongoing or developing disease processes. This work lays the groundwork for studies that examine the potential role of other components in cigarettes or e-cigs as it relates to neurological disorders.