Exposure to electronic cigarette vapor exacerbates ischemic loss of tryptophan/tyrosine hydroxylase-positive neurons in the rat brain

Abstract

The rising use of nicotine-containing electronic cigarettes (EC) underscores the need to understand their impact on stroke outcomes. Our previous work showed that 16 days of EC exposure worsened post-stroke cognitive decline in young rats of both sexes, likely through altered brain energy metabolism. However, its downstream effects on neurotransmitters (NT) remain unclear. This study investigates how EC exposure affects NT metabolism and its consequences on ischemic injury. Adult Sprague-Dawley rats were exposed to air or EC for 16 nights. One cohort underwent global metabolomic profiling of cortical tissue. A second cohort received transient middle cerebral artery occlusion (tMCAO; 90 min) or sham surgery, then survived 1 day before cortical samples were collected for tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH) protein analysis. A third cohort underwent the same exposures but survived for 21 days, after which brains were collected for immunohistochemical analysis. Three brain sections per animal were labeled with anti-TPH or anti-TH antibodies to visualize TPH-positive and TH-positive neurons in the dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and substantia nigra (SN). Neuron counts were estimated using a brightfield stereoscope and StereoInvestigator software. EC-exposed females showed reduced post-stroke neuronal survival across all brain regions compared to air-exposed females; this effect was not observed in males. Metabolomics revealed significant (p ≤ 0.05) increases in phenylalanine and tryptophan metabolites, along with mixed changes in tyrosine metabolites. A marked reduction in TH- and TPH-positive neurons was also observed post-tMCAO in EC-exposed females. These NT alterations, more prominent in females, may contribute to EC-related cognitive decline and depressive symptoms, underscoring the need for further investigation into EC's impact on ischemic brain injury.

Keywords: Dorsal raphe nucleus; Metabolomics; Neurotransmitters; Substantia nigra; Tryptophan hydroxylase; Tyrosine hydroxylase; Ventral tegmental area.