Electronic Cigarette Vape Exposure Exacerbates Post-Ischemic Outcomes in Female but Not in Male Rats

Abstract

Background: Nicotine-containing electronic cigarette (EC) vaping has become popular worldwide, and our understanding of the effects of vaping on stroke outcomes is elusive. Using a rat model of transient middle cerebral artery occlusion, the current exploratory study aims to evaluate the sex-dependent effects of EC exposure on brain energy metabolism and stroke outcomes.

Methods: Adult Sprague-Dawley rats of both sexes were randomly assigned to air/EC vapor (5% nicotine Juul pods) exposure for 16 nights, followed by randomization into 3 cohorts. The first cohort underwent exposure to air/EC preceding randomization to transient middle cerebral artery occlusion (90 minutes) or sham surgery, followed by survival for 21 days. During the survival period, rats underwent sensorimotor and Morris water maze testing. Subsequently, brains were collected for histopathology. A second cohort was exposed to air/EC after which brains were collected for unbiased metabolomics analysis. The third cohort of animals was exposed to air/EC and received transient middle cerebral artery occlusion/sham surgery, and brain tissue was collected 24 hours later for biochemical analysis.

Results: In females, EC significantly increased (P<0.05) infarct volumes by 94% as compared with air-exposed rats, 165±50 mm³ in EC-exposed rats, and 85±29 mm³ in air-exposed rats, respectively, while in males such a difference was not apparent. Morris water maze data showed significant deficits in spatial learning and working memory in the EC sham or transient middle cerebral artery occlusion groups compared with the respective air groups in rats of both sexes (P<0.05). Thirty-two metabolites of carbohydrate, glycolysis, tricarboxylic acid cycle, and lipid metabolism were significantly altered (P≤0.05) due to EC, 23 of which were specific for females. Steady-state protein levels of hexokinase significantly decreased (P<0.05) in EC-exposed females; however, these changes were not seen in males.

Conclusions: Even brief EC exposure over 2 weeks impacts brain energy metabolism, exacerbates infarction, and worsens poststroke cognitive deficits in working memory more in female than male rats.

Keywords: Morris water maze test; electronic nicotine delivery systems; glycolysis; icotine; vaping.

Figure 1:

(A) Experimental design including the EC exposure paradigm delineated in the triangular diagram underneath the exposure period. (B) Cotinine levels in the brain of rats exposed to air or EC. (C, E) Representative images of cerebral blood flow (CBF) in the ipsilateral cortex before, during, and after tMCAO. (D, F) Ipsilateral CBF (presented as percentage of baseline) in tMCAO groups significantly decreases (p<0.05) during the occlusion period compared to baseline in both (D) female and (F) male cohorts. (n=7, “*” = p<0.05).

Figure 2:

(A) Infarct frequency heatmaps show the area of infarcted tissue as denoted by color intensity. The third heatmap in each row shows the within-sex significant difference (p<0.05) in infarct frequency between air-exposed and EC-exposed cohorts as determined by a Fisher’s exact test and color intensity represents 1.0 – p-value. (B) EC significantly exacerbates (p<0.05) ischemic infarction after stroke in female animals and (C) shows a trend of increased infarct volume in male animals as depicted in the shown graphs of infarct volume (mm³), respectively. (D-E) Cylinder test quantification is presented as asymmetry index and shows that at 7 DPI, tMCAO-exposed females exhibit significantly impaired sensorimotor function. EC- and tMCAO-exposed females do not significantly improve asymmetry by 21 DPI. tMCAO-exposed males exhibit significantly impaired sensorimotor function at 7 DPI. Air- and EC-exposed males significantly improve asymmetry index at 21 DPI compared to 7 DPI. (n=7 except for infarct volume of air- and tMCAO-exposed females where n=6; “*” = p<0.05, “$” = p<0.05 compared to baseline).

Figure 3:

(A) Line graph depicts latency (in seconds) to find the hidden platform. EC exposure did not reduce latency to find the hidden platform over the period of four day trial period, suggesting spatial learning deficits in animals of both sexes. (B) EC-exposed female rats spent significantly less time in the goal quadrant on probe trial testing (Day 5) than air-exposed, though only in sham cohort. EC-exposure did not significantly affect time spent in the goal quadrant in male rats. (C) Female and male rats exposed to EC did not significantly decrease the latency to find the platform in the second of paired trials in both sham and tMCAO cohorts, suggesting EC-induced impairment of working memory. (D) tMCAO in EC-exposed females significantly decreased CA1 neuronal survival compared to respective air-exposed rats, but not in males. (n=7 and “*” = p<0.05).

Figure 4:

(A) Experimental design indicates that brains were collected for metabolomics 16-days after air/EC exposure. (B) Glycolytic and carbohydrate metabolite levels significantly altered due to EC exposure include 10 metabolites only changing in females and 5 metabolites only changing in male animals compared to respective air-exposed group. (C) The TCA cycle metabolites fumarate and acetylphosphate were only significantly altered (p<0.05) in female rats exposed to EC compared to female air controls, and no TCA metabolite levels were significantly altered due to EC exposure in males. Metabolite levels are depicted as means with error bars denoting SD and individual values represented as dots. (n=6, “*” = p<0.05, “#” = p<0.05 compared to same exposure condition of the other sex).

Figure 5:

Lipid metabolite levels are differentially altered in female and male animals exposed to EC compared to respective air-exposed animals. 11 metabolites were significantly changed in only female EC-exposed rats and 4 metabolites were significantly altered in only in EC-exposed male rats. Metabolite levels are depicted as means with error bars denoting SD and individual values represented as dots. (n=6, “*” = p<0.05, “#” = p<0.05 compared to same exposure condition of the other sex).

Figure 6:

(A) Experimental design for Western blotting of glycolytic and phospholipid enzymes. (B) Graphs depict Western blot analysis of key enzymes. EC exposure significantly decreased levels of hexokinase in the cortex of female rats. (C) EC exposure did not change protein levels of glycolytic or lipid metabolism enzymes in the cortex of male rats. (n=6, “*” = p<0.05).