The facial area is an important site of autonomic reflexes involving parasympathetic and sympathetic nervous systems in the human and these reflexes have trigeminal fibers as afferent branches. Therefore, stimulation of specific facial regions may induce reflex with certain important implications (Brunelli et al., 2012). Recently, we showed that, in normotensive anesthetized rats and in normotensive humans, a mandibular extension (ME) induced a reduction in blood pressure (BP) (Lapi et al., 2013, 2014; Del Seppia et al., 2016, 2017). Moreover, in normotensive rats, this effect was abolished by bilateral peripheral trigeminal section, thus indicating a fundamental role of trigeminal nerve in the hypotensive ME-induced response (Lapi et al., 2013). Furthermore, we have also shown that two periods of MEs in the normotensive rat prolongs the effects of a single ME on BP, heart rate (HR) and vasodilation, modulating the pial arteriolar tone by the increase of endothelial activity (Lapi et al., 2014). Therefore, ME might represent a procedure useful in the control of the systemic BP and, consequently, of the organ perfusion, in particular of the brain and vasculature in cardiovascular diseases, such as hypertension.

Since renin-angiotensin system (RAS) is known to play an important role in the pathophysiology of hypertension, in the present study, we evaluated the gene and protein modulation of RAS after two periods of MEs in a spontaneous hypertensive rat (SHR) model. Furthermore, we evaluated gene expression of RAS components in a group of normotensive (NT) rats undergoing ME procedure, in order to verify that observations on SHRs were effectively related to the hypertensive condition. The molecular analysis was carried out on brain parietal area, where principal trigeminal afferent fibers project, and on brain frontal area, less directly involved in trigeminal nerve functions. It is commonly believed that the effects of AngII (Angiotensin II) on brain depend mostly on AT1R receptor stimulation and that excessive brain AT1R activity correlates with hypertension, heart failure, brain ischemia and more in general, with neurodegenerative disorders (Jackson et al., 2018). However, in the last decades, protective action of the RAS in the nervous system have been investigated and AT2R and MAS1 receptor have been demonstrated to counteract, in most cases, AT1R inflammatory, fibrotic and neurogenerative effects (Horiuchi and Mogi, 2015, Prestes et al., 2017). MAS1 receptor is the main mediator of Ang(1-7) peptide, synthesized by angiotensin-converting-enzyme 2 (ACE2) through the hydrolysis of AngII (Villela et al., 2015). Ang(1-7) has been proven to have important vasodilator, diuretic and natriuretic effects, inhibiting cell growth and norepinephrine release and increasing bradykinin effect (Santos et al., 2003). Aim of the present study is to focus on the efficacy of a repeated ME protocol on main physiological parameters (BP and HR) and the possible effects on RAS deleterious (ACE/AngII/AT1R) and protective (ACE2/Ang(1-7)/MAS1 or ACE/AngII/AT2R) arms in two different rat cortical regions.

In the present study we evaluated the effects of a repeated ME procedure on modulation of main physiological parameters (BP and HR) and of principal components of RAS in SHRs, focusing primarily on the response in cerebral cortex. Previous studies in normotensive rats, investigating arteriolar tone modifications, have shown that the stimulation of trigeminal nerve by ME procedure determines a prolonged hypotensive effect accompanied by initial rapid vasoconstriction, followed by a prolonged vasodilation y in the pial microcirculation (Lapi et al., 2017). Furthermore, our studies on humans demonstrated that the submaximal mouth opening for a relatively brief time (10 min) was followed by a prolonged hypotensive and bradycardic effect on normotensive volunteers (Del Seppia et al., 2016, 2017). Due to the important role of RAS in the pathophysiology of hypertension, we focused on the gene expression and protein levels of RAS components during ME protocol at cortex level in SHRs and in NT rats.

It is commonly believed that AngII acts via its receptor AT1R to increase BP, stimulating sympathetic effects and vasopressin secretion (Jancovski et al., 2013). However, besides the deleterious involvement of ACE/AngII/AT1R system in BP alteration, it is conceivable that other components of RAS, collectively known as “protective RAS” may be engaged to counteract the hypertensive drive. As far as we know, protective RAS consists of two major arms: ACE/AngII/AT2R and ACE2/Ang(1-7)/MAS1 (Steckelings and Unger, 2012). In the brain, there is some evidence that AT2R and MAS1 not only functionally oppose to AT1R in BP control but also have unique effects that lie outside of AT1R signaling (Horiuchi and Mogi, 2015; Prestes et al., 2017) and that mostly regard cognitive functions (Sakata et al., 2009), cell survival, antioxidant and anti-inflammatory properties (de Kloet et al., 2017). Moreover, until recently, the potential central nervous system-mediated effects of Ang II/AT2R and Ang(1-7)/MAS1 in BP regulation and hypertension have been understudied, and still not well understood (de Kloet et al., 2017). More recently, a better understanding of the distribution of AT2R in the brain was supported by fluorescence in situ hybridization studies, which provided a discrete view of the cellular localization of AT2R in this organ (de Kloet et al., 2016). From information available on AT1R and AT2R expression, data in mouse indicated an overlap between the two AngII receptors (Lenkei et al., 1997) and it has been hypothesized that direct antagonistic effects of the two receptors in the same cells use different mechanisms to interfere in the same pathways (Matsuura et al., 2005). In hypertensive rats, the supplemental action of MAS1 receptor and involvement of Ang(1-7) is believed to be crucial in the compensatory activity aiming to restore a normal BP (Lucas et al., 2015). Conversely, in primary murine astrocytes cultures, has been observed that the presence of AT2R is required for Ang(1-7) activity and, reversely, MAS1 is required for beneficial effects of AT2R, suggesting a synergistic interplay between receptors and ligands (Leonhardt et al., 2017). It is known from the literature that SHR hypertensive phenotype in the brain is greatly sustained by an alteration of a balance in ACE/ACE2, and that AngII synthesis is favored with respect to Ang(1-7) generation (Gowrisankar and Clark, 2016). Specifically, in our SHR model, two hypotensive ME procedures were performed, as previously described in normotensive rats (Lapi et al., 2017). These results are in accordance with those obtained in normotensive rats in which a repeated stimulation induced a decline in BP by nearly 20 mmHg (Lapi et al., 2017), and suggest that the hypotensive effect is even greater (about 28 mmHg, Figure 2B) and more prolonged in hypertensive animals. At molecular level, AT1R and ACE gene expression were significantly reduced only in parietal area while they did not change in frontal region. Differently, AT2R and ACE2 gene expression did not vary both in parietal and frontal regions (Figures 3A,C). Furthermore, in the two cortical areas analyzed, an opposite response was observed for Mas1 receptor gene expression, since it was significantly reduced after the hypotensive 2-ME procedure in the parietal region (Figure 3A) and greatly increased in the frontal area (Figure 3C). Since both AT1R and AT2R protein levels significantly decreased in frontal area (Figure 4B), a possible post-translational mechanism, inducing the inhibition of AngII receptors can be hypothesized. Moreover, at protein level, MAS1 was significantly reduced in the parietal area (Figure 4A) of 2-ME rats, in accord with gene expression, and no significant changes were observed in the frontal region (Figure 4B). In accord with what previously discussed about MAS1 role in hypotensive response, our data suggest that, in our model of SHRs undergoing two MEs, the involvement and augmentation of expression of Mas1 at frontal cortex level, might play an important role in the restoration of BP and cardiac parameters in SHRs. Differently, a significant augmentation of AT1R and ACE gene expression observed after two MEs in the parietal area (Figure 3A), might be attributed to a feedback response to the hypotensive effect induced by ME procedure, and possible reduction of AngII levels. Furthermore, in order to verify that observations in SHRs were effectively related to the hypertensive state, we performed an analogous investigation in a group of NT rats undergoing ME procedure. Interestingly, the gene expression analysis in NT rats revealed a very different pattern for the five studied genes both in parietal and frontal areas, when compared to SHRs. The complete non-detectability of AT1R in both areas of NT rats is in accord with its role as AngII principal receptor in pathological settings, mainly in hypertension. In parietal area, both AT2R and Mas1 were significantly increased in NT SO with respect to NT 2-ME, suggesting their important role in the preservation of normotensive conditions. Furthermore, in parietal area, ACE and ACE2 resulted N/D in NT SO rats, whereas there was some expression of these genes after 2-ME procedure (Figure 3B). Differently, in the frontal area of NT rats, no difference was observed in the expression of all genes tested between SO and 2-ME rats (Figure 3D). Therefore, (1) in the two brain areas studied RAS gene expression pattern is very different in SHRs with respect to NT rats and (2) 2-ME procedure supports RAS protective arm effects in chronic hypertensive conditions but not in NT subjects.

Our data indicate that in our SHR model a double ME procedure induces a hypotensive response and that a modulation of gene expression and protein levels of main RAS components occur in cerebral cortex regions. In particular, in a translational perspective, a very interesting aspect of our study is that the hypotensive effects induced by the double ME persisted for the entire post-treatment observation period of 240 min. In future studies, it will be interesting to evaluate the effects of repetitions also in consecutive days in order to analyze how far the effects of repetitive ME will drive the hypotensive response. Moreover, to evaluate the effects of the same procedures after the treatment with specific inhibitors of ACE2 or MAS1 will add important information on involvement of the different RAS metabolites analyzed. In conclusion, on the basis of previous data on rats and humans and the present data on SHRs and NT rats, it is possible to speculate that possible new procedures, implying repetitive MEs, may find a clinical application as non-pharmacological ancillary aid in acute or chronic treatment of arterial hypertension.

LS and RS conceived the project. CC, DL, and GF performed the experiments. LS, CDS, SB, AC, GI, and RS contributed to discussions regarding the project. LS, CDS, and RS analyzed the data, wrote and revised the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.