Simultaneous nicotine and oral contraceptive exposure alters brain energy metabolism and exacerbates ischemic stroke injury in female rats

Abstract

Smoking-derived nicotine (N) and oral contraceptives (OC) synergistically exacerbate ischemic brain damage in the females and underlying mechanisms remain elusive. Our published study showed that N toxicity is exacerbated by OC via altered mitochondrial function owing to a defect in the activity of cytochrome c oxidase. Here, we investigated the global metabolomic profile of brains of adolescent female Sprague-Dawley rats exposed to N ± OC. Rats were randomly exposed to saline or N + /−OC for 16–21 days followed by random allocation into two cohorts. One cohort underwent transient middle cerebral artery occlusion and histopathology was performed 30 days later. From the second cohort, cortical tissues were collected for an unbiased global metabolomic profile. Pathway enrichment analysis showed significant decrease in glucose, glucose 6-phosphate and fructose-6-phosphate, along with a significant increase in pyruvate in the N + /−OC exposed groups when compared to saline (p < 0.05), suggesting alterations in the glycolytic pathway which were confirmed by Western blot analyses of glycolytic enzymes. Infarct volume quantification showed a significant increase following N alone or N + OC as compared to saline control. Because glucose metabolism is critical for brain physiology, altered glycolysis deteriorates neural function, thus exacerbating ischemic brain damage.

Introduction

Stroke remains a leading cause of death, and women have higher lifetime risk of stroke, an increased mortality rate from stroke, and a higher tendency for recurrent strokes than men.^1–3 Strikingly, there are studies that clearly observe more damaging effects in women who combine oral contraceptive/ hormone replacement therapy (OC/HRT) and cigarette smoking. This also points to the fact that: (1) OC/HRT and cigarette smoking have synergistic deleterious effects on a women’s brain,⁴ and (2) a gender non-specific risk factor. Cigarette smoking-derived nicotine (N) – have unique effects on the female brain that need to be identified and targeted in order to reduce consequences of stroke in women. An estimated 22% of American women are exposed to nicotine through smoking, which often begins in adolescence.⁵ Adolescence is also the critical period of life when young women initiate sexual activities and are more likely to use OC. Although it has been known for some time that long-term use of OC and nicotine causes an increased risk of peripheral thrombus formation,^6–13 the exact mechanism by which a combination of OC and nicotine (N + OC) causes cerebral ischemia is not clear.

Our published study shows that nicotine toxicity is aggravated by OC via altered mitochondrial function and increased production of reactive oxygen species in the hippocampus of adult female rats.¹⁴ The mitochondrial dysfunction involved a deficiency in the enzymatic activity of the terminal enzyme of the electron transport chain (cytochrome c oxidase or complex IV). However, how this mitochondrial dysfunction affects overall brain metabolism remains to be investigated. Understanding changes in brain metabolism at different ages after N + OC exposure is important, as neurons are considered to produce energy via an oxidative metabolism. Alterations in the metabolic pathways related to energy production will have detrimental consequences for neuronal survival. Therefore, it becomes imperative to understand what are the changes, and what are the basis for these brain metabolic switches and how are they are affected by exposure to substances such as nicotine. In the current study, we hypothesize that defects in the respiratory chain caused by N + OC will have defined metabolic consequences, which can increase susceptibility to ischemic stroke in adolescent female rats. We also hypothesize that adolescent brain is more susceptible to nicotine toxicity and ischemic damage following N/N + OC exposures as compared to adult female rats.

Materials and methods

Animals

All experimental procedures were carried out as per the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and in accordance with the protocols approved by the Animal Care and Use Committee of the University of Miami. Results are reported according to the Animal Research: Reporting in Vivo Experiments guidelines to the best of our knowledge.

We obtained adolescent (6 weeks old) and adult (14 weeks old) Sprague-Dawley rats. The adolescent Sprague-Dawley rats at six weeks (their estrous cycles have been established by this time) are capable of reproduction.¹⁵ We examined their vaginal smears daily to monitor the estrous.^4,16 Rats showing at least three consecutive normal periods (four days) of estrous cycles were used for experiments as described in our earlier publications.^17,18 Following establishment of normal estrous cycle, rats of both ages (n = 8) were randomly allocated to following experimental conditions.

Nicotine exposure

Rats were anesthetized (Isoflurane), an area on the back (3 cm × 6 cm) is shaved and an incision was made to permit subcutaneous implantation of an osmotic minipump (type 2ML2, Alzet Corp., Palo Alto, CA). The incision was sutured and the rats were placed in their cages for recovery. The pump delivered a fixed and continuous dose of nicotine hydrogen tartrate (4.5 mg/kg/day, equivalent to 1.5 mg/kg/day free base) or saline throughout the 16–21 days.¹⁴ The rats were monitored every day for the duration of nicotine treatment.

Oral contraceptive treatment

As described in our previous publication, rats were treated with an OC for 16–21 days (∼4 estrous cycles).¹⁴ The dose was scaled to mimic a woman’s daily OC dose based on a diet of 1800 cal/days. Because rats need an average of approximately 32 cal/100 g of body weight/day, a 250–290 g rat needed an OC dose based on an intake of 80–93 cal/days. The rats were given OC by an oral gavage for three consecutive days and placebo (placebo tablet given along with OC pills; administered orally) on the fourth day based on the 4-day estrous cycle of rat, so as to resemble OC administration in women.¹⁹

Transient middle cerebral artery occlusion and infarct volume

An investigator blinded to the experimental treatments performed transient middle cerebral artery occlusion (tMCAO) surgical and behavioral procedures. Nicotine, OC, N + OC or saline-treated rats were exposed to ischemic stroke (90 min) using an intraluminal suture inserted past the internal carotid artery to occlude the middle cerebral artery as described previously.²⁰ In parallel, we also performed sham surgery on saline-treated rats. During this sham procedure, rats were exposed to anesthesia for a period similar to that of the tMCAO group. During the surgical procedure of tMCAO or sham, physiological parameters including pCO₂, pO₂, pH, HCO₃ and arterial blood pressure were maintained within normal limits prior to and after tMCAO (Supplemental Table 1). We also monitored regional cerebral blood flow by Laser Doppler Flowmetry (LDF, Perimed Inc.). A sudden and sharp decrease in the LDF signal indicated a successful MCA occlusion. Rats that had less than 70% drop in LDF signal on suture insertion and premature mortality during tMCAO were excluded from the study. The suture was gently withdrawn after the 90-min occlusion period. Body and head temperatures were maintained at 37°C ± 0.2 throughout the experiment with assistance of lamps placed above the animal’s body and head.

One month after tMCAO surgery, rats were perfused with saline, then with FAM (a mixture of formaldehyde, glacial acetic acid, and methanol; 10:10:80), and brains were prepared, sectioned and stained to obtain infarct volumes. The details of sample preparation and procedures remain same as described in our earlier publication.²¹ Brains were then embedded with paraffin and 10 µm thick sections were obtained. The method of infarct volume measurement is described in our earlier publications.^22,23 Briefly, the same nine coronal sections were selected for every animal (Bregma levels 5.2, 2.7, 1.2, −0.3, −1.3, −1.8, −3.8, −5, −7.3). Infarcted area was then measured on each section using MCID software. Infarct volume was then determined similar to the previously used methods.^22,23

Additionally, a standardized neurobehavioral test battery was also conducted as described previously,²⁰ which includes tests for postural reflex, sensorimotor integration and proprioception. The neurobehavioral test was performed at 1, 7, 15 and 30 days after induction of tMCAO.

Global metabolomic analysis

At the end of N and N + OC treatments, cortical tissue was harvested (n = 8 for each group), immediately frozen in liquid nitrogen, and sent to Metabolon Inc. for processing. They obtained a global metabolomic profile using a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a Thermo Scientific Q-Exactive high resolution/accurate mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer. Samples were processed and aliquots analyzed for acidic positive ion conditions with specific chromatographic solvents optimized to detect hydrophobic and hydrophilic compounds. Sample aliquots were also analyzed for negative ion conditions in a separate C18 platform and HILIC columns (Waters). Compounds were identified by comparison to library entries of more than 3300 purified standards of known retention time/index (RI), mass to charge ration (m/z) and chromatography data. Data were curated with multiple procedures and proprietary software to ensure high quality data and compound identification. Metabolite levels were quantified by using the area under the peak and normalized by protein. Bioinformatic analysis was also performed by Metabolon Inc. Two types of analysis were performed: significance test and classification analysis using ArrayStudio and Program R or JMP. The analysis included three-way ANOVA, calculating p and q-values (false discovery rate). Data obtained are represented as significant-fold change in a table format or as scaled intensity in box plot format for each group. Circles in the graph represent extreme data points; error bars represent maximal and minimum distribution; horizontal line on the box represents the median value; box length represents the limit of upper and limit of lower quartiles; and the “ + ” sign inside the box represents the mean value.

Western blotting

Cortical tissues were homogenized; protein content was determined and proteins were separated by 12% stain-free SDS-PAGE as described.³⁰ Proteins were transferred to polyvinylidene difluoride (PVDF) membrane and incubated with primary antibodies against anti-hexokinase 1 (1:1000; cat # 2024; Cell signaling), anti-lactate dehydrogenase A (LDHA) (1:1000; Cat # 3582/2012; Cell Signaling), anti-phospho-LDHA (1:1000; cat # 8176; Cell signaling), anti-pyruvate kinase (1:1000; cat # 4053; Cell signaling), anti-phospho-PKM2 (1:1000; cat # 3827; Cell signaling), and anti-β-actin (1:5000; Sigma). All data were normalized to β-actin (monoclonal; 1:1000; Sigma). Immunoblot images were digitized and subjected to densitometric analysis.¹⁸

Hexokinase enzymatic activity

In the hexokinase assay protocol, glucose is converted to glucose-6-phosphate by hexokinase. The glucose-6-phosphate is then oxidized by glucose-6-phosphate dehydrogenase to form NADH. To determine HK activity, we followed the production of NADH spectrophotometrically at 340 nm.²⁴

Statistical analysis

An investigator blinded to the experimental conditions performed Western blot, enzyme activity and stroke infarct volume data analysis. Rats that had more than a 70% drop in cerebral blood flow during tMCAO were included in the final analysis. Mean differences were analyzed using one-way ANOVA with Bonferroni’s post hoc test for multiple group comparisons. Neurological scores were analyzed using Kruskal–Wallis ANOVA. Data are presented as mean ± standard deviation.

Results

NOC exacerbates post-tMCAO infarction in adolescent and adult rat brains

The results described above showed the impact of N and N + OC on energy metabolism. Therefore, we investigated the synergistic effect of N + OC on ischemic brain damage in adolescent female rats. We quantified infarct volume as an index of focal cerebral ischemic damage. Figure 1(a) shows a significant increase in infarct volume following N alone or in the N + OC groups. In adolesent rats, N alone or combination of N + OC exposures followed by ischemia resulted in 78 ± 6 mm³ (n = 6) and 108 ± 8 mm³ (n = 7), respectively, which was significantly higher than 33 ± 3 mm³ (n = 7; p < 0.05) in the saline or 34 ± 9 mm³ (n = 5) in the OC-treated groups. These results showed that the synergistic deleterious effect of N + OC exacerbates post-tMCAO infarct volume as compared to N alone in adolescent female rats. Similarly, N exposure to adult female rats followed by ischemic insult resulted in 50.38 ± 15 mm³ (n = 6), while N + OC caused 83.46 ± 6 mm³ (n = 7) of infarct volume. These results showed that the synergistic deleterious effect of N + OC exacerbates post-tMCAO infarct volume as compared to N alone in both adolescent and adult female rats.

Figure 1.

N+OC exacerbates post-tMCAO infarction in rat brains. (a) Infarct volume measurements observed in saline, N, OC and N + OC exposed adolescent female rats at one-month recovery from tMCAO. There were significant differences observed between groups (one way ANOVA, Bonferroni post hoc test). (b and c) Neurological deficit (ND) scores assessment at 1, 7, 15 and 30 days post-tMCAO in different experimental groups of adolescent and adult rats, respectively.

Total neurological score ranged from a normal score of 0 to a maximal possible score of 12. Results demonstrated significant lower improvement in neurological score in the N or N + OC exposed adolescent and adult rats as compared to respective, saline groups (Figure 1(b) and (c)). Adolescent and adult rats demonstrated a month survival rate of 22% (7/32) and 19% (6/32), respectively. All mortality except for four animals (one adolescent saline and two adolescent OC-exposed and one adult OC-exposed) occurred within four days of tMCAO surgery.

Since the intensity of ischemic brain damage was higher in adolescent as compared to adult female N/N + OC exposed rats, for the subsequent metabolomics study we present data on adolescent rats and corresponding adult rats data are presented in supplemental section.

Nicotine alone or in combination with OC impaired mitochondrial CIV activity

In our published study, we reported significant decreases in the activity of hippocampal mitochondrial CIV in N + OC-exposed groups as compared with the rest of the experimental groups. Here we confirmed that reduced CIV was not only confined to the hippocampus but also was reduced in the cortex. Figure 2 depicts reduced CIV activity in the cortex of adolescent rats exposed to saline, N, OC and N + OC, suggesting that cortical mitochondrial function is also compromised owing to nicotine toxicity.

Figure 2.

N+OC decreases cortical mitochondrial Complex-IV activity. Graph represents data of complex IV activity measured by spectrophotometer in cortical homogenates obtained from different experimental groups.

Accumulation of nicotine and its metabolites in cortical tissue

The global metabolomic profile showed that both nicotine and its metabolites cotinine and nornicotine accumulated in cortical brain tissue following continuous nicotine exposure in N and N + OC treatment groups in relation to controls, confirming that nicotine is reaching the brain following osmotic pump-mediated delivery (Figure 3 and Supplemental Table 2a). Accumulation of nornicotine and cotinine following nicotine administration suggests that these metabolites might play a contributory role in the neuropharmacological effects of nicotine (Figure 3(c) to (e)). Furthermore, nicotine accrual was more pronounced in N + OC exposure, suggesting that OC may elevate the neuro-stimulatory actions of nicotine in the brain. The levels of nicotine and its metabolites were higher in N and N + OC-treated adolescent rats compared to adults (Supplemental Table 2b and c), suggesting that the ability of the brain to accumulate nicotine and its metabolites might decrease with age.

Figure 3.

Accumulation of tobacco metabolites in the cortex of adolescent female rats. (a) Depicts nicotine metabolic pathway. (b) Table shows fold-change of nicotine metabolites, red box indicates increase. (c–e) Box plot shows nicotine metabolites-fold change in the cortex of saline, N, OC and N + OC exposed rats.

N + OC alters glycolysis and TCA cycle metabolites in the brain

Tight regulation of glucose metabolism is critical for brain physiology. The global metabolomic profile revealed significant changes in many glycolytic intermediates in N, OC and N + OC-treated rats in relation to saline treatment, suggesting altered cortical energetics in response to treatments (Figure 4). Figure 4(b) and (c) and Supplemental Table 3 show significant-fold change decrease in the levels of glucose, glucose 6-phosphate and fructose-6-phosphate along with a significant increase in the glycolytic end-product pyruvate in N and N + OC exposed rats in comparison to saline controls. These results suggest two possible scenarios: (1) that there is a relative increase in conversion of glucose use in response to treatment-mediated energy demand, or (2) that glycolysis is downregulated and accumulation of pyruvate is observed because there is either a decreased conversion to lactate (following N exposure) or an inefficient mitochondrial oxidative phosphorylation system that cannot drive the TCA cycle. The last scenario is supported by the increased levels of citrate, succinate and aconitate (Figure 5(b) to (e)), although the increased input of pyruvate into the TCA also could lead to higher levels of citrate and aconitase. In rats treated with OC, there were no changes in glucose and glucose 6 phosphate but there were significant increases in the levels of fructose 1,6 diphosphate, 3-phosphoglycerate, phosphoenolpyruvate and pyruvate (Figure 4(b) and Supplemental Table 4).

Figure 4.

N+OC alters glycolysis metabolites in the cortex of adolescent female rats. (a) Depicts glycolytic pathway, green font indicates decrease and red font indicates increase in the particular metabolite. (b) Table shows fold-change of glycolysis metabolites, green box indicates decrease, red indicates increase and light green indicates narrowly missed statistical cutoff for significance 0.05<p < 0.10. (c–e) Box plot shows alterations in glucose, glucose-6-phosphate and pyruvate-fold change in the cortex of saline, N, OC and N + OC exposed rats.

Figure 5.

N+OC alters TCA cycle metabolites in the cortex of adolescent female rats. (a) Depicts TCA cycle, red font indicates increase in the particular metabolite. (b) Table shows fold-change of TCA cycle metabolites; red box indicates increase and pink indicates narrowly missed statistical cutoff for significance 0.05<p < 0.10. (c–g) Box plot shows alterations in citrate, aconitate, succinate, NAD⁺ and FAD⁺ fold change in the cortex of saline, N, OC and N + OC exposed rats.

Concomitant increases in biochemicals related to energy metabolism including creatine phosphate, nicotinamide adenine dinucleotide (NAD⁺) and FAD with N and/or N + OC treatment in the brain could possibly be an indication that metabolic flow may be specifically shunted toward energy production, leading to altered energy metabolism in response to N and/or N + OC exposure (Figure 5(f) and (g) and Supplemental Table 4).

N + OC alters hexokinase enzyme activity in the brain

Because we observed alterations in glycolysis metabolites in rats exposed to N and N + OC, we analyzed the enzymatic activity of hexokinase (HK), one of the key regulatory enzymes of this pathway. HK catalyzes the first step of glycolysis, phosphorylating glucose into glucose 6 phosphate (Figure 6(a)). HK has a relatively low Km for glucose and it is regulated by end product inhibition. Figure 6(b) shows that exposure to nicotine and N + OC decreased the enzymatic activity of HK and the decrease was statistically significant for the N + OC exposed rats compared to saline controls. Western blot analysis revealed the decreased HK activity was due to a decrease in the steady-state levels of this enzyme (Figure 6(c) and (d)). The decreased HK activity supports the notion of a decreased energy metabolism in N and N + OC exposed rats.

Figure 6.

N+OC alters hexokinase enzyme activity in the cortex of adolescent female rats. (a) Depicts conversion of glucose to glucose-6-phosphate mediated by hexokinase enzyme. (b) Represents data of hexokinase enzyme activity measured by spectrophotometer in cortical tissue from different experimental groups. (c) Immunoblots shows steady-state protein levels of hexokinase in the cortex of saline, N, OC and N + OC exposed rats. Beta-actin was the loading control. (d) Western blot analysis demonstrates a significant reduction in the steady state protein level of hexokinase in the cortex of in N and N+OC exposed rats as compared to saline.

N + OC-induced metabolic changes correlate with the protein levels of glycolytic enzymes

Because the levels of HK were altered, we also investigated the steady-state levels of other glycolytic enzymes. Figure 6 shows decreased levels of enolase, pyruvate kinase isoform m2 (PKM2), pPKM2, lactate dehydrogenase A (LDHA) and pLDHA in N or N + OC-treated rats compared to saline controls. N + OC-instigated decrease in the three enzymes was statistically significant (Figure 7). Decreased levels of glycolytic enzymes in N and N + OC exposed rats are in agreement with decreased glycolysis intermediates observed in the global metabolomic profile.

Figure 7.

N+OC induced metabolic changes correlate with the protein levels of glycolytic enzymes. (a) Immunoblots show steady-state protein levels of enolase, PKM2, and LDHA in the cortex of saline, N, OC and N + OC exposed rats. Beta-actin was used as loading control. (b-f) Western blot analysis demonstrates a significant reduction in the steady state protein level of enolase, PKM2, LDHA, phospho-PKM2 and phospho-LDHA in the cortex of in N and N+OC exposed rats as compared to saline.

Discussion

Women who smoke cigarettes using oral contraceptives are at increased risk for and higher severity of stroke compared to nonsmoking women who use OC.^25–29 In the current study, using an adolescent and adult female rat model of N exposure through cigarette smoking and typical OC regimens of women, we showed that the simultaneous exposure to N and OC caused (1) significantly greater cortical infarction and (2) severe ischemic brain damage compared to OC or nicotine alone in adolescent as compared to adult female rats. This study demonstrates that N + OC altered the global metabolomic profile of adolescent female brain. Specifically, the pathway enrichment analysis showed significant changes in energy metabolism (glycolysis and TCA cycle) in the brains of rats exposed to N + /−OC in relation to saline treatment. Western blot analyses of glycolytic enzymes support the observed metabolic changes. Because glucose metabolism is critical for brain physiology, altered glycolysis deteriorates neural function, thus exacerbating ischemic brain damage.

In the female brain, N + OC exposure significantly reduced the levels of some mitochondria-encoded proteins, and affected the activity of one of the complexes of the electron transport chain that, in turn, reduced mitochondrial function and increased the production of reactive oxygen species. Our findings showed that nicotine toxicity is aggravated by OC via an inhibitory effect on mitochondrial function.^14,30 In the current study, we demonstrated N or N + OC significantly reduced cortical mitochondrial CIV enzyme activity. This becomes particularly important in that neurons derive most of their energy by oxidative metabolism.³¹ The energetic metabolism of the brain is a complicated and still not completely understood process. A metabolic trafficking between astrocytes and neurons has been proposed (the astrocyte-neuron-lactate shuttle) in which glutamate released from synaptic terminals is taken up by the astrocytes which process in turn stimulates glucose consumption.³² During glycolysis, the astrocytes produce lactate, which is exported and taken up by the neurons via monocarboxylic transporters that convert it to energy by oxidative phosphorylation.^33,34 This has recently been challenged, and albeit glucose is considered the main energy substrate, the brain has the capacity to use alternative substrates.^35–37 Indeed, in the rodent brain, there is a switch on metabolic fuels during development. During gestation, the embryonic brain uses glucose as main fuel source, while during lactation it uses a combination of glucose and ketone bodies, and after weaning the brain relies more on glucose (review³⁷). These changes in metabolic fuels continue from the adolescent into the adult brain and whether they are altered by other factors as substance abuse, such as nicotine, requires further investigation.

Hexokinase, phosphofructokinase and pyruvate kinase are the three regulatory enzymes in the glycolysis pathway. They catalyze irreversible steps in the pathway and the enzymatic activity is subjected to allosteric or post-translational modifications. The fact that we observed reduced enzymatic activity of HK and reduced steady-state levels of HK and PKM2 indicates that N and N + OC induces changes in the energy-generating pathway at the gene expression level and most likely will affect the gene expression of other cellular pathways. It is known that changes in cellular metabolism also alter epigenomic marks; therefore, the impact in gene expression profiles is even larger.

Nicotine is pleiotropic in nature, and therefore the observed alterations in energy metabolism in mitochondrial function could induce long-term consequences for cerebral blood flow,³⁸ edema³⁹ and inflammation.⁴⁰ In a recently published study using an in vitro model of ischemia in organotypic slice cultures, we demonstrated that chronic nicotine exposure exacerbated neuronal death after oxygen–glucose deprivation (OGD) via activation of the inflammasome.⁴⁰ Furthermore, inhibition of nicotine-induced inflammasome activation improves post-OGD neuronal survival, suggested that most of the impairment is in neurons and not glia. Inflammasome is a key component of innate immune response and they are either members of the NLR family or members of the pyrin and HIN domain-containing (PYHIN) family.⁴¹ The NLRP3 inflammasome, which regulates the secretion of interleukin (IL)-1β, is considered to be a sensor of altered metabolic homeostasis.⁴² On the contrary, metabolic signals from mitochondria are known to modulate inflammatory response. Mitochondrion-derived danger-associated molecular patterns, such as mtDNA, directly confer inflammatory changes in microglial and neuronal cells.⁴³ Emerging evidence from various laboratories suggests that the mitochondrial oxidative phosphorylation system is a primary site of action during acute inflammation and that it is a central determinant of ischemic damage. Microglia and monocyte-derived macrophages largely play protective functions in ischemic brain injury. The macrophages are highly specialized in sensing the microenvironment and modify their properties accordingly.^44,45 Although it is very complex to categorize/classify the macrophages, they exist in polarized forms: a pro-inflammatory (M1) and an anti-inflammatory/pro-resolving (M2) profile. It is now proposed that the M1 macrophages rely mainly on glycolysis, while M2 cells are more dependent on oxidative phosphorylation.⁴⁶ Altered immunometabolism may hinder the M1 to M2 switch in the brain of N + OC exposed rats, which may be responsible for exacerbating ischemic brain damage observed in current study. Since the observed metabolic alterations in the brain after N or N + OC exposure were assessed in whole tissue, our study will not be able to distinguish if the changes occur in neurons or in glia: this remains as one of the main caveats of our study. Nevertheless, the results of the current study provide a basic footprint for future analysis that could be done in neuronal or glial cultures. Importantly, discerning the exact effects of N + /−OC on overall brain metabolism and the molecular mechanisms affecting mitochondrial function opens a new window for future therapeutic intervention.

Altered glucose metabolism and altered mitochondrial electron transport chain leads to decreased ATP levels and deterioration of neuronal function which, in turn, results in poor outcomes after an ischemic insult as we observed in adolescent rats treated with N + OC. To complicate things further, recent studies showed that the expression of monocarboxylic transporters is modulated by peripheral metabolism, suggesting that the adult brain is sensitive and can adapt to changes in the overall metabolism in response to changes in diet and/or genetic composition, as shown in mice.⁴⁷ Therefore, it becomes imperative to understand what the changes are and what the bases for these brain metabolic switches are, and how they are affected by exposure to substances such as nicotine. We believe that defects in the respiratory chain caused by N + OC will have defined metabolic consequences, which can increase susceptibility to other conditions/diseases.

We observed higher levels of nicotine and its metabolites in N and N + OC exposed rats (Supplemental Table 2b and c), which is consistent with published literature.⁴⁸ Because young accumulate more nicotine than adults, this may have even more devastating consequences as smoking-related mortality accounts for an average loss of 14 years from a woman’s life.⁴⁸ In recent years, smokers trying to quench their habit have switched to electronic nicotine delivery systems (e-Cigarettes). In contrast to regular cigarettes that contain a complex chemical mixture containing 4800 different compounds, e-cigarettes deliver dosages of nicotine based on the smokers’ needs. The e-cigarettes are marketed as a “healthy tool for smoking cessation” and claim that it enables smokers to obtain their nicotine without the carcinogens found in cigarette smoke. E-cigarettes advertisements target young women, especially adolescent girls who are a relatively new marketing group. Ours is the first study of this nature to lay the foundation for translation to the human population. This makes the proposed research high impact and timely. The results of our research show direct effects of nicotine on the brain in OC-using women that are responsible for the increased severity of brain ischemia. Therefore, the outcomes of our research will be helpful in educating everyone, along with providing scientific evidence for a legislative impetus to raise the e-cigarette age to 21 years in order to avoid lasting effects on teenage brains.