Short-term exposure to JUUL electronic cigarettes can worsen ischemic stroke outcome

Average plasma nicotine concentrations in JUUL and TS-exposed mice were 22.5 ± 6.68 ng/ml and 74.76 ± 5.95 ng/ml, respectively, whereas those of cotinine were 42.58 ± 5.2 ng/ml and 194.7 ± 24.42 ng/ml, respectively. Plasma concentration of nicotine was significantly higher in TS-exposed mice (P < 0.0001) compared to JUUL-exposed mice (Fig. 1A). Similarly, TS-exposed mice had a higher plasma level of cotinine (P < 0.0001) than JUUL-exposed mice (Fig. 1A). Further, when we measured the ratio of plasma cotinine to nicotine, we found that the ratio was significantly decreased in JUUL-exposed mice (1.25) (P < 0.05) than in TS-exposed mice (1.88) (Fig. 1B). This ratio characterizes nicotine metabolism to cotinine in mice after JUUL/TS exposure.

We measured the weight of the mice after 14 days of JUUL or TS exposure and 24 h after MCAO (Fig. 2A, B). We found that JUUL (P < 0.0001) or TS (P < 0.0001) exposure for 14 days drastically reduced the weight of the mice compared to the control (Fig. 2A). TS-exposed mice also had significant weight reduction (P < 0.001) compared to JUUL-exposed mice. MCAO caused weight reduction in all groups, but no significant weight difference was observed among control, JUUL, and TS-exposed mice 24 h after MCAO. The locomotor activity of the mice was measured by open field test after 14 days of JUUL or TS exposure and 24 h after MCAO (Fig. 2C, D). JUUL-exposed mice had a non-significant increase in percent baseline activity change compared to control, while TS-exposed mice showed significant hyperactivity (P < 0.05) compared to control mice (Fig. 2C). No significant difference was observed in the total distance traveled by the mice 24 h after MCAO among the 3 groups (Fig. 2D). TS exposure significantly affected the weight and locomotor activity of the mice, while the effects of JUUL exposure were to a lesser extent.

We did 2,3,5-tripheyltetrazolium chloride (TTC) staining 24 h after MCAO to evaluate brain injury in the mice (Fig. 3A–C). JUUL (P < 0.01) and TS-exposed (P < 0.05) mice had significantly increased brain infarct area compared to control mice (Fig. 3A, B). There was also an increase in brain swelling ratio in TS-exposed mice (P < 0.05) compared to control (Fig. 3A, C). Further, the neurological score was significantly worsened in TS-exposed mice (P < 0.01 vs. control) 24 h after MCAO (Fig. 3A, D). Although JUUL exposure increased brain infarct area, it did not significantly affect brain swelling and neurological function after MCAO.

Plasma IL-6 and thrombomodulin levels were measured in mice by ELISA after 14 days of JUUL or TS exposure and 24 h after MCAO (Fig. 4A, B). IL-6 level, a key inflammatory marker, was higher in control MCAO (P < 0.01 vs. control, P < 0.05 vs. JUUL, and P < 0.05 vs. TS) and TS MCAO (P < 0.01 vs. control, P < 0.05 vs. JUUL, and P < 0.01 vs. TS) groups (Fig. 4A). In contrast, the IL-6 level in JUUL MCAO was not significant. In contrast, plasma thrombomodulin level was lower in control MCAO (P < 0.05 vs. control), JUUL MCAO (P < 0.0001 vs. control, P < 0.001 vs. JUUL, and P < 0.01 vs. TS), and TS MCAO (P < 0.05 vs. control) groups (Fig. 4B). JUUL MCAO had a more significant effect than TS MCAO for plasma thrombomodulin, indicating more coagulation potential.

We measured the expression of BBB tight junction (TJ) proteins (claudin-5 and occludin) and TJ-associated protein ZO-1 by western blot and immunofluorescence in the normoxic and contralateral and ipsilateral brain hemispheres 24 h after MCAO (Figs. 5A–L, 6A–C, 7A–F). JUUL and TS exposure had a comparable damaging effect on the TJ-associated protein ZO-1. JUUL (P < 0.01) and TS (P < 0.01)- exposed mice had a significant reduction of ZO-1 expression after MCAO in the contralateral brain hemisphere compared to control (Fig. 5A, C) in the western blot. There was no substantial change in normoxic and ipsilateral brain ZO-1 levels among the groups (Fig. 5A, B, D). This data is supported by immunofluorescence studies which showed JUUL (P < 0.05) and TS (P < 0.05)-exposure reduces ZO-1 expression in the contralateral brain hemisphere (Fig. 6A, B) compared to control while there was a non-significant downregulation of ZO-1 in the ipsilateral hemisphere (Fig. 6A, C). In the western blot, claudin-5 expression in the contralateral brain hemisphere of TS-exposed mice was reduced (P < 0.05) compared to control (Fig. 5E, G), while JUUL exposure had no significant effect. Also, no significant change in claudin-5 expression in the normoxic and ipsilateral brain regions was observed among the groups (Fig. 5E, F, H). In immunofluorescence studies, claudin-5 expression showed a decreasing trend in the contralateral hemisphere with JUUL and TS exposure, but it was not statistically significant (Fig. 7D, E). No change in claudin-5 expression was observed in the ipsilateral hemisphere (Fig. 7D, F). Occludin expression was also significantly reduced in the contralateral brain regions of JUUL (P < 0.05 vs. control) and TS (P < 0.01 vs. control)-exposed mice (Fig. 5I, K) in western blot experiments. Further, JUUL-exposed mice had decreased occludin expression (P < 0.05) in the ipsilateral brain region compared to control (Fig. 5I, L), with no significant change observed with TS exposure. Also, no change in occludin expression in the normoxic brains was observed (Fig. 5I, J). Immunofluorescence studies strongly support these results, where JUUL (P < 0.01) and TS (P < 0.05) exposure decreased occludin expression in the contralateral hemisphere (Fig. 7A, B). In the ipsilateral hemisphere, only JUUL exposure (P < 0.05) decreased occludin expression (Fig. 7A, C). JUUL exposure had a more significant effect on occludin expression in the ischemic brain than TS.

We then measured brain expression of the inflammatory marker ICAM-1 and the antioxidant marker Nrf2 by western blot in the normoxic and, contralateral & ipsilateral brain hemispheres 24 h after MCAO (Fig. 8A–H). No significant change was observed in normoxic brain Nrf2 expression among the groups (Fig. 8A, B). JUUL (P < 0.05) and TS (P < 0.05)-exposed mice had a significant reduction of Nrf2 expression after MCAO in the contralateral brain hemisphere compared to control (Fig. 8A, C). Interestingly, immunofluorescence studies supported the western blot findings in the contralateral hemisphere, showing a significant reduction of Nrf2 expression by JUUL (P < 0.05) and TS (P < 0.001) exposure (Fig. 6D, E). No significant difference was observed in ipsilateral Nrf2 expression by western blot (Fig. 8A, D). However, immunofluorescent studies showed a significant reduction of Nrf2 by TS (P < 0.0001) and JUUL (P < 0.001) exposure in the ipsilateral hemisphere (Fig. 6D, F). Western blot results showed that ICAM-1 expression was significantly increased in the ipsilateral brain regions of TS (P < 0.05 vs. control)-exposed mice (Fig. 8E, H). There was no significant change in ICAM-1 expression in the group’s normoxic and contralateral brain regions (Fig. 8E–G).

All studies were approved by the IACUC of Texas Tech University Health Sciences Center, Lubbock, Texas (IACUC protocol# 20026). All experiments were performed in accordance with relevant guidelines and regulations. This study was not pre-registered, and no randomization/blinding was performed. Male C57BL/6 mice (Charles River Laboratories, Inc., Wilmington, MA) were kept under standardized light and dark conditions (12 h), humidity (70%), and temperature (22 °C). They were given ad libitum access to food and water.

The behavior of the animals was monitored every day to minimize animal suffering. We applied the following exclusion criteria: severe weight loss, infections, or significant behavioral deficits (decreased mobility, seizures, lethargy). No animal was excluded from this study. The study is reported following ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. The research design is depicted as a flow diagram in Fig. 9.

Mice were exposed (via direct inhalation) to JUUL e-Cig vapor (30 mg/ml nicotine) or 3R4F standardized research cigarettes (9.4 mg tar and 0.726 mg nicotine/cigarette equivalent to full flavor commercial products) mixed with oxygenated air or oxygenated air alone, 6 cycles/day for 14 days. A modified CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) standard smoking protocol adapted to study JUUL exposure (27.5 ml puff depth volume, 3 s puff duration, 2 puffs per 60 s, 32 puffs/cycle) and a modified CIR (Canadian Intense Regimen) standard smoking protocol (27.5 ml puff depth volume, 2 s puff duration, 2 puffs per 60 s, 32 puffs/cycle) to study TS exposure were followed in the laboratory. E-Cig vapor/TS was generated using a Single Cigarette Smoking Machines (SCSM, CH Technologies Inc., Westwood, NJ, USA) following a previously published method [12, 15]. These methods were followed to mimic the smoking behavior of a human chronic and heavy smoker/vaper and yield plasma levels of cotinine (43 and 195 ng/ml for JUUL and TS, respectively) which is in the range of blood cotinine levels found in other preclinical models of chronic TS/e-Cig exposure [23, 27, 34]. The smoking exposure was done between 9 am to 2 pm.

The plasma concentration of nicotine and its principal metabolite cotinine were measured from the 14 days JUUL/TS-exposed mice by LCMS/MS analysis using Cotinine-d3 (MilliporeSigma, St. Louis, MO, USA) as internal standard (IS) following a previously published method [23]. In brief, samples were prepared by protein precipitation of 25 µl mouse plasma using acetonitrile at a 1:8 ratio. The mass spectrometer was operated in positive polarity under the multiple reaction monitoring mode using the electrospray ionization technique. The transitions of m/z 163.2 → 132.1, 177.2 → 98.0, and 180.2 → 101.2 were used to measure the nicotine, cotinine, and IS, respectively. The elution of nicotine (MilliporeSigma), cotinine (MilliporeSigma), and IS were at 1.89, 1.77, and 1.76 min, respectively. This was achieved with a mobile gradient phase consisting of 5 mM ammonium bicarbonate, acetonitrile, and methanol (3:1, v/v) at a 0.3 ml/min flow rate on a Kinetex EVO C18 column (Phenomenex, Torrance, CA, USA).

Open field test was performed according to our previously published study [20, 75]. This test evaluated the locomotor activity of the JUUL/TS-exposed or control mice with or without stroke. We used Versamax software (Accuscan Instruments., Columbus, OH) to automatically calculate the activity of the animals (total distance traveled). Briefly, mice were introduced to a 16″ × 16″ unobstructed glass chamber. They were monitored and recorded for 1 h. The first 10 min of 1 h was excluded as the acclimatization period. All experiments were performed between 8 am, and 11 am.

Transient Middle cerebral artery occlusion (tMCAO) surgery was performed in mice (24–28 g) as previously reported [75, 76], using a Zeiss OP pico I surgical microscope (Carl Zeiss GmbH, Jena, Germany). Mice were anesthetized with 4% and maintained at 1.5% isoflurane in N₂O/O₂ mixture (70/30) using a SurgiVet Vaporizer (Smith Medical North America, Waukesha, WI, USA). Continuous blood flow was measured with a Laser Doppler probe (Moor Instruments, Wilmington, DE, USA). The probe was placed on the skull directly above the left MCA region (1 mm posterior and 3 mm lateral to the Bregma). Body temperature was maintained at 37 °C and controlled by a thermostatic blanket (TC-1000 Temperature Controller, CWE, USA). After aseptic preparation with betadine, a 1.5 cm long incision was made on the neck midline. The left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully isolated from surrounding tissue. After CCA was occluded, a micro clip was placed on the ICA, and the ECA was ligated and coagulated. A small incision on the ECA was made to introduce a 6–0 nylon microfilament with a round tip (0.20–0.25 mm), and it was gradually inserted until it blocked the MCA bifurcation. A decrease in blood flow of 80% from baseline was considered a successful occlusion. After 30 min of occlusion, the nylon filament was carefully removed to restore blood flow, leading to reperfusion. An increase of 70% or more of the blood flow during occlusion was considered successful reperfusion.

The neurological score was evaluated in mice 24 h after reperfusion. A four-point scale was utilized for this purpose [77]. A score of 0 indicated no neurological deficits, 1 indicated mild focal neurological deficit (animal showed forelimb flexion), 2 indicated moderate focal deficit (decreased resistance to lateral push and forelimb flexion), while 3 indicated severe focal deficit (animal showed all previous deficits plus circling).