Cerebral Cortical Vasodilation via Nicotinic Receptors by Heated Tobacco Product Aerosol Extract in Rats

Cigarette smoking increases the risk of developing many diseases such as lung cancer or cardiovascular disease and associated event, such as myocardial infarction or stroke in smokers and those exposed to second-hand smoke [1‒4]. The use of novel tobacco products instead of conventional combustion cigarettes has increased in recent years [5]. Those using novel tobacco products such as heated tobacco products (HTPs) inhale aerosols generated by heating tobacco leaves instead of smoke generated by burning it [6]. There exist very little data on the biological effects of HTPs, including comparison with conventional combustible cigarettes from human or experimental animals, even of the acute effects [7].

The nicotine contained in tobacco is a major contributor to the immediate increase in regional cerebral blood flow (rCBF) caused by smoking [8, 9]. Intravenous injection of nicotine increases rCBF in humans [8]. Of the various subtypes of nicotinic acetylcholine receptors (nAChRs), the α4β2 and α7 subtypes are the most abundant and widespread in the mammalian brain, including the neocortex [10]. The important role of α4β2 type nAChRs in increasing prefrontal cerebral blood flow during cognitive task has been demonstrated in humans [11]. Studies using anesthetized animals have shown that intravenous injection of a low dose of nicotine increases rCBF in the cortex, independent of systemic arterial pressure [12‒14]. A study using selective agonists showed that a selective α7 nAChR agonist (i.v.) produced no detectable changes in cerebral blood volume in the cortex, whereas a selective α4β2 agonist (i.v.) induced an increase in cerebral blood volume in anesthetized rats [15]. Another study using selective antagonists showed that the nicotine-induced increase in cerebral blood flow in the cortex is abolished by a selective α4β2 antagonist, while it is not affected by a selective α7 antagonist [16]. These studies suggest that the nicotine-induced increases in blood flow in the cerebral cortex are mediated by activation of α4β2-type nAChRs, but not of α7-type nAChRs.

Based on the abovementioned background studies, we hypothesized that the novel nicotine-containing HTPs may produce an increase in cortical rCBF due to activation of α4β2-type nAChRs. Therefore, this study aimed to clarify the acute effects of novel HTPs on cortical rCBF in anesthetized rats. First, we compared the effects of intravenous injections of smoke extract prepared from conventional combustion cigarettes with those of aerosol extract prepared from an HTP on cortical rCBF, and second, we examined the contribution of the nAChR α4β2 subtype to rCBF responses to injection of those extracts.

The experiments in this study were conducted on 11 male Fischer rats (330–410 g; 4–10 months old). Our research was conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments (established by the Science Council of Japan in 2006), and the study protocol was approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute for Geriatrics and Gerontology (Animal Ethics Committee: approval number 21018-3). This study is reported in accordance with ARRIVE guidelines.

The rats were anesthetized with subcutaneous injection of urethane (1.4 g/kg), after initial inhalation of 4.2% sevoflurane for approximately 5 min. Respiration was maintained using an artificial respirator (SN-480-7, Shinano, Tokyo, Japan) via a tracheal cannula. The end-tidal CO₂ concentration was monitored using a gas monitor (Capnostream™ 35, Oridion Medical, Jerusalem, Israel) and was maintained at 3.0%–4.0% by controlling respiratory volume and frequency. Arterial blood pressure was measured with a pressure transducer (TP-400T, Nihon Kohden, Tokyo, Japan) through a catheter inserted into a femoral artery. Body temperature was measured rectally and continuously using a thermistor and maintained at approximately 37.5°C using a body temperature control system (ATC-101B-RS-S1, Unique Medical, Tokyo). Anesthesia was sustained with additional urethane doses (100 mg/kg, i.v. via a catheter inserted into a femoral vein, or s.c.) when necessary. This was determined by monitoring body movement, blood pressure stability, and respiratory movement. The analog arterial blood pressure signal was converted to a digital signal on a desktop computer using an analog to digital converter (Micro 1401 mkII, Cambridge Electronic Design, Cambridge, UK) and Spike 2 software (Spike 2, Cambridge, UK).

rCBF was measured using laser speckle contrast imaging, as previously described [17‒19]. The animals were mounted on a stereotactic instrument (SR-5R-HT, Narishige, Co., Ltd., Tokyo, Japan) in a prone position. The skull was thinned with a dental drill to allow the visualization of the rCBF through the bone, without damaging the meninges or causing leakage of the cerebrospinal fluid. The skull surface was covered with mineral oil. For laser speckle contrast imaging, a Moor full-field perfusion-imaging device consisting of an infrared laser diode (785 nm wavelength) and a charge-coupled device camera (Moor Instruments, Devon, UK) was fixed. The zoom lens was adjusted to produce an image of the entire dorsal surface of the brain from the most anterior part of the olfactory bulbs to the most posterior aspect of the occipital cortex, and the polarizer lens was carefully adjusted to minimize speckle reflection. The viewing field covered approximately 300 mm² (20 × 15 mm) with a matrix of 152 × 113 pixels, providing an approximate resolution of 132 µm/pixel. The images were sampled at 25 Hz with exposure time of 4 ms. On-line traces were generated from a rectangular region of interest positioned in the left frontal cortex (AP = 1.0–3.5 mm, L = 1.0–3.0 mm). On-line averaging generated one mean image every 1 min. Continual acquisition of these images throughout the trial provided 25–40 images over 25–40 min.

To analyze spatial changes in the blood flow, the baseline image acquired immediately before administration of drugs (two kinds of extracts or nicotine) was then subtracted from the other images to assess the relative blood flow changes. To quantify the temporal blood flow changes (in arbitrary units: AU), time courses were extracted from eight regions of interest (ROIs); six 1.5 mm-diameter circular ROIs positioned bilaterally to avoid visible blood vessels in the area of the frontal (AP = 1.0–4.0 mm, L = 1.0–4.0 mm), parietal (AP = 0 to −3.0 mm, L = 2.0–5.0 mm), and occipital cortices (AP = −5.0 to −8.0 mm, L = 1.5–4.5 mm) [20, 21] and two oval ROIs (1.0 mm-width, 1.5 mm-height) positioned bilaterally in the area of the olfactory bulb (AP = 6.5–8.0 mm, L = 0.5–1.5 mm). The rCBF changes induced by administration of two kinds of extracts or nicotine were expressed relative (%) to the baseline signal immediately before the administration.

In this study, reference cigarette 1R6F (University of Kentucky, Center for Tobacco Reference Products, Lexington, KY, USA), and commercial HTP Ploom S (Japan Tobacco Inc., Tokyo, Japan) [22] were used. Methods for preparing smoke extract of the reference combustion cigarette and aerosol extract of the HTP in dimethyl sulfoxide (DMSO) solution have been described previously [23]. The cigarette smoke extract and HTP aerosol extract contained nicotine at concentration of 2.21 mg/mL and 2.92 mg/mL, respectively, when determined by a previously published method using a gas chromatograph equipped with a flame ionization detector (Agilent 7890, Agilent Technologies, Santa Clara, CA, USA) [24]. The two kinds of extracts were diluted in saline and were used at doses equivalent to 30 μg nicotine/kg body weight (calculated as the free base). At nicotine doses of 30 μg/kg, the final concentrations of DMSO in the diluted solutions of cigarette smoke extract and HTP aerosol extract were 4.5% and 3.4%, respectively. Nicotine (Tokyo Kasei Kogyo, Tokyo, Japan) was diluted in saline and used at doses of 3, 10, 30, and 100 μg/kg body weight (calculated as the free base). These solutions were then injected slowly (over approximately 1 min) into the femoral vein of the rat.

The α4β2-preferring nAChR antagonist dihydro-β-erythroidine hydrobromide (DHβE, Tocris, Bristol, UK, 5 mg/kg) was dissolved in saline and administered intravenously as reported previously [16, 25]. We investigated the effects of i.v. administration of 3, 10, 30, and 100 μg nicotine/kg on cortical rCBF and blood pressure in five rats. In each rat, doses of 3, 10, and 30 µg/kg were administered in a randomized order, while a dose of 100 µg/kg was always administered last. We waited for about 10–30 min between each injection, or waited until all effects of the drugs had disappeared. We used other six rats to investigate the effects of i.v. administration of 30 µg nicotine/kg, and doses of cigarette smoke extract and HTP aerosol extract, equivalent to 30 µg nicotine/kg, on cortical rCBF and blood pressure. The order of administration of cigarette smoke extract, HTP aerosol extract, and nicotine to each rat was randomized. The effects of DHβE administration were examined in four of these six animals.

All values are presented as mean ± SEM, unless otherwise stated. Data analysis was done using Prism 9 (GraphPad Software Inc., San Diego, CA, USA). Temporal changes in the rCBF and the mean arterial pressure, and comparisons of rCBF changes obtained from different cortical areas or produced by different drugs (cigarette smoke extract, HTP aerosol extract, and nicotine) were assessed by the Friedman test followed by Dunn’s multiple comparison test. Comparison of rCBF changes obtained from the left and right hemispheres were assessed by Wilcoxon matched-pairs signed-rank test. A p value of <0.05 was considered statistically significant.

To determine the nicotine equivalent dose suitable for comparing cigarette smoke extract and HTP aerosol extract, responses of frontal lobe blood flow and mean blood pressure following i.v. injection of nicotine at doses of 3, 10, 30, and 100 µg/kg were examined in five rats. Frontal cortical blood flow was dose-dependently increased by 10–100 µg nicotine/kg, while mean arterial pressure was increased at only a dose of 100 µg nicotine/kg (online suppl. Fig. 1; for all online suppl. material, see https://doi.org/10.1159/000541726). Therefore, we chose a nicotine dose of 30 µg/kg, which significantly increased frontal cortical blood flow without changing blood pressure for further experiments.

Speckle imaging (Fig. 1a(A, B), b(A, B)) and extraction of blood flow signal from the left frontal cortex (Fig. 1a(C), b(C), c) showed that i.v. injection of cigarette smoke extract and HTP aerosol extract (at a dose of 30 µg nicotine/kg) increased cortical cerebral blood flow over a similar time course without changing mean arterial pressure. The vehicle of extracts (5% DMSO solution) did not affect the cortical blood flow (Fig. 1d) and mean arterial pressure. In both cigarette smoke extract and HTP aerosol extract, the increase in frontal cortex blood flow usually started within 50 s after the start of the injection, appeared significant 1–2 min after the injection (Fig. 2). The significant increases in blood flow lasted for 8–9 min, and gradually returned to the preinjection level. The maximum blood flow response to cigarette smoke extract and HTP aerosol extract at 3–4 min was 146 ± 8% (n = 6) and 153 ± 7% (n = 6), respectively (Fig. 2). Neither cigarette smoke extract nor HTP aerosol extract changed the mean arterial pressure (Fig. 2). I.v. administration of 30 µg/kg of nicotine itself also increased frontal cortical blood flow, with a maximal response of approximately 145 ± 7% (n = 11) at 3–4 min (online suppl. Fig. 2). The magnitudes of increases in frontal cortical blood flow induced by cigarette smoke extract, HTP aerosol extract, and nicotine itself were equivalent, and there were no significant differences between these drugs.

Blood flow signals in different regions of the cerebral cortex (frontal cortex, parietal cortex, occipital cortex, and olfactory bulb) of both hemispheres were analyzed in six rats. Fig. 3a shows the time course of HTP aerosol extract-induced change in blood flow in each region of the right hemisphere. The magnitude and time course of the blood flow increase were similar in the frontal, parietal, and occipital cortices. By contrast, blood flow did not significantly change in the olfactory bulb (Fig. 3a). The blood flow response pattern was the same in the left hemisphere. Similar to HTP aerosol extract, cigarette smoke extract and nicotine at a dose of 30 µg nicotine/kg increased blood flow in the frontal, parietal, and occipital cortices but not in the olfactory bulb. Concerning the magnitudes of the blood flow responses to cigarette smoke extract (Fig. 3b), HTP aerosol extract (Fig. 3c), and nicotine (online suppl. Fig. 3) in the frontal, parietal, occipital cortices, there were no significant differences between left and right hemispheres, and between cortices (frontal, parietal, and occipital).

The contribution of α4β2-like nicotinic receptors on the cortical cerebral blood flow response was tested in a total of four rats. An α4β2-preferring nicotinic receptor antagonist, DHβE (5 mg/kg), was shown to abolish increased frontal cortex blood flow induced by HTP aerosol extract, and cigarette smoke extract (both at a dose equivalent to 30 µg nicotine/kg) in all four rats tested (Fig. 4).

The present study showed that in anesthetized rats, intravenous injection of smoke extract of conventional combustion cigarette increases cortical rCBF without changes in blood pressure. Furthermore, this study is the first to demonstrate that injection of aerosol extract of novel HTP increases cortical rCBF without changes in blood pressure. Moreover, regarding the mechanism of the rCBF increase, the contribution of α4β2-like neuronal nAChRs in the brain was clarified.

The present results showed that increases in cortical rCBF induced by HTP aerosol extract and cigarette smoke extract were widespread in the cortex, i.e., in the bilateral frontal, parietal, occipital cortices. The characteristics of the increased cortical rCBF response (magnitude, time course, brain area) induced by these two extracts (both at a dose equivalent to 30 µg nicotine/kg) were similar to those of the increased cortical rCBF response to nicotine administration (30 µg/kg), and all were abolished by α4β2-preferring nAChR antagonist DHβE. These results suggest that the major acute effect of these two extracts on cortical vessels, i.e., vasodilation resulting in elevated cortical rCBF, is mediated by nicotine-induced α4β2-nAChR activation, setting aside toxic non-nicotine constituents of these two extracts as potential mediators of this effect [26‒29].

Previous studies in anesthetized rats showed that the nicotine-induced cortical rCBF elevation was not affected by methyllycaconitine, an α7-selective nicotinic antagonist, whereas it was completely abolished by dihydro-β-ethythroidine, an α4β2-preferring nicotinic antagonist [16]. Bilateral lesioning of the nucleus basalis of Meynert attenuated the nicotine-induced rCBF elevation by approximately 50% but significant elevation of cortical rCBF remained [14]. Furthermore, the nicotine-induced cortical rCBF elevation was inhibited by administration of a nitric oxide synthase inhibitor [30]. Therefore, it is suggested that cortical rCBF elevation induced by intravenous nicotine injection is due to nitric oxide-mediated vasodilation following activation of nAChRs, mainly of the α4β2-like subtype, on both cholinergic neurons in the basal forebrain (nucleus basalis of Meynert) and the cortex [14, 16, 30]. The mechanism underlying the increase in cortical rCBF in response to injection of nicotine-containing HTP aerosol extract and cigarette smoke extract in the present study may be the same. Electrical stimulation of the nucleus basalis of Meynert in the basal forebrain produces a significant increase in cortical rCBF without influencing the diameter of pial arteries at the cortical surface [31, 32]. On the other hand, intravenous administration of nicotine causes significant increases in the diameter of pial arteries at the cortical surface [33]. Therefore, vasodilation of both pial arteries and parenchymal blood vessels may have contributed to the increase in cortical rCBF induced by intravenous administration of HTP aerosol extract and cigarette smoke extract in the present study. The mechanism of α4β2 nAChR-mediated increase in cortical cerebral blood flow in anesthetized animals may be related to the mechanism of immediate cerebral blood flow increase induced by cigarette smoking and intravenous nicotine infusion in humans [8, 9].

The plasma nicotine concentration reaches 10–50 ng/mL after smoking a cigarette, and it reaches averaged 23 ± 14.7 ng/mL (mean ± SD) after i.v. administration of 25 μg/kg nicotine in healthy adult smokers [34]. In rats, the plasma nicotine concentration reaches approximately 1460 ng/mL after i.v. administration of 1 mg/kg nicotine [35]. Based on this report [35], we can estimate that i.v. administration of 30 μg/kg nicotine (the dose used in the present study) may increase the plasma nicotine concentration to about 44 ng/mL, which is equivalent to the plasma nicotine concentration after smoking a cigarette in humans [34]. Compared with conventional combustion cigarettes, the maximum plasma nicotine concentration is low (approximately 75% or less than half) after the use of HTPs, although the HTP devices are different from the one used in the present study [36, 37]. Therefore, cortical rCBF elevation induced by the nicotine intake by HTP use may be smaller than that induced by the nicotine intake by cigarette smoking.

Our previous study investigated the effect of aging on the nicotine-induced cortical rCBF elevation using male animals of older age. In rats 23–26 months old (approximately 2 years old), a i.v. administration of 30 μg/kg nicotine increased cortical rCBF to a similar extent as in the younger rats (3–10 months old). In contrast, in 32- to 36-month old rats (approximately 3 years old), nicotine at 30 μg/kg had no significant effect on the cortical rCBF [14]. The variation of 4–10 months of male rats used in this study is within the range of adult rats (3–10 months) of previous study [14].

The limitation of this study is that only males were examined, and sex differences were not considered. Since the cerebral vasculature is a target tissue for sex steroids [38], there may be sex differences in the cortical rCBF response induced by HTP and conventional combustion cigarettes. In the future, it is necessary to investigate the sex difference in cortical rCBF responses.

A previous report showed that the acute nicotine-induced cortical rCBF elevation was reduced after chronic subcutaneous nicotine treatment in rats [16]. Compared to nonsmokers, rCBF values were reduced in chronic smokers [39, 40]. Moreover, compared to never-smokers, former-smokers (long-term cessation of smoking) had lower CBF values [41]. Further studies are needed to investigate the chronic use of HTPs on rCBF compared to combustible cigarette smoking. In conclusion, as a biological effect of novel HTPs, this study showed that when intravenously administered in anesthetized rats, these aerosol extracts acutely produce an increase in cortical rCBF through activation of α4β2-like neuronal nAChRs.

This study protocol was reviewed and approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute for Geriatrics and Gerontology, Approval No. [21018-3].

The authors have no conflicts of interest to declare.

This study was funded by Japan Tobacco Inc. The funder had no role in the design, data collection, data analysis, and reporting of this study.

Sae Uchida: conceptualization, funding acquisition, investigation, data curation, formal analysis, writing – original draft, writing – review editing, and supervision. Jura Moriya, Daichi Morihara, and Mayura Shimura: investigation, data curation, and writing – review editing. Fusako Kagitani: investigation, data curation, formal analysis, and writing – review editing.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.