**1. Introduction**

Hypertension is one of the most important risk factors of cardiovascular disease, and despite the current treatment options, a substantial portion of the population still have uncontrolled or suboptimal controlled blood pressure (BP) [1]. Additionally, the aetiology of hypertension remains unknown in most cases. Sympathetic nervous system (SNS) has an integral role in the regulation of heart rate and contractility, vascular tone and fluid volume. SNS hyperactivation leads to retention of salt and/or water and increases cardiac output and peripheral resistance [2]. In addition, elevated SNS also participates in vasculokar remodelling related to smooth-muscle hypertrophy and fibrosis [3], contributing to the development and/or the maintenance of hypertension through an increase in peripheral resistance [4–6]. Recent advances have led to the understanding that hypertension may have a developmental origin. It is now well accepted that the foetus can adapt to adverse intrauterine conditions promoting physiological alterations in foetal development to ensure

**Citation:** Vieira-Rocha, M.S.; Sousa, J.B.; Rodríguez-Rodríguez, P.; Arribas, S.M.; Diniz, C. Elevated Vascular Sympathetic Neurotransmission and Remodelling Is a Common Feature in a Rat Model of Foetal Programming of Hypertension and SHR. *Biomedicines* **2022**, *10*, 1902. https://doi.org/ 10.3390/biomedicines10081902

Academic Editor: Ivana Vanˇeˇcková

Received: 1 July 2022 Accepted: 4 August 2022 Published: 5 August 2022

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survival [7]. Such alterations, later in life, may increase the susceptibility to develop hypertension and cardiometabolic diseases, in a process named as foetal programming [8–10]. Several adverse factors during intrauterine life have been demonstrated to contribute to inadequate foetal development and foetal programming. The most important ones are malnutrition [9,11], oxygen deprivation [12–14], placental insufficiency [15], exposure to excess of glucocorticoids [16–18], toxic substances (alcohol, tobacco) [19–21] and environmental pollutants [22]. The mechanisms underlying an offspring's predisposition to develop hypertension in adulthood have not been completely addressed. Nevertheless, implication of increased oxidative stress [23,24], alterations in the glucocorticoid axis [16,25–27] or activation of the renin-angiotensin system (RAS) [28–30] have been suggested. Some of these alterations might be mediated by epigenetic modulation of genes implicated in cardiovascular control [24,31–34] and/or alterations in renal or vascular autonomic functions [14,26,35–38]. In the context of foetal programming hypertension (FPH), the contribution of the peripheral sympathetic nervous system is still not completely understood [39]. The spontaneously hypertensive rats (SHR), developed by Okamoto and Aoki 1963 [40] is a well-established model, which resembles essential hypertension in humans [41]. Among other common features, the SHR exhibits alterations in RAS [42], sympathetic hyperinnervation [43] and resistance artery remodelling [44], features also present in human essential hypertension. SHR and rat models of FPH also share some similarities regarding blood pressure development, i.e., the sexual dimorphism and the time course of development [45,46].

In the current work, we hypothesize that hypertension of developmental origin may share features with SHR regarding sympathetic hyperinnervation, which may contribute to vascular remodelling and hypertension development. Our aims were to evaluate, in mesenteric arteries from SHR and from a rat model of FPH induced by maternal undernutrition during gestation (MUN), the (i) sympathetic innervation, (ii) sympathetic activation and (iii) vascular remodelling.

#### **2. Materials and Methods**

#### *2.1. Animals*

Sprague–Dawley, Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats from the colony maintained at the animal house facility of the Universidad Autónoma de Madrid were used. All experimental procedures were approved by the Ethics Review Board of Universidad Autónoma de Madrid and Comunidad Autónoma de Madrid (CEI63-1112-A097 and PROEX 04/19) according to the Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised in 1996), the Spanish legislation (RD 1201/2005) and the Directive 2010/63/EU on the protection of animals used for scientific purposes. The rats were housed in buckets 36.5/21.5/18.5 cm (length/width/height) on aspen wood bedding, under controlled conditions of 22 ◦C, 40% relative humidity and 12/12 light/dark photoperiod. The animal health monitoring indicated that they were free from pathogens that may interact with any of the parameters studied. The ARRIVE Guidelines were followed for reporting in vivo experiments [47].

#### 2.1.1. Experimental Model of FPH

FPH model based on global maternal nutrient restriction was induced as previously described [46,48]. Two study groups were established using the Sprague–Dawley strain: a CONTROL group, with ad libitum feeding throughout pregnancy and lactation, and a group with intake restriction during part of gestation (maternal undernutrition model, MUN). This last group of rats had ad libitum diet during the first half of the gestation (from day 1 to 10), and then they were fed with 50% of the intake of a pregnant rat (from day 11 until delivery). The maximum daily intake of rat chow was previously determined in a group of pregnant rats as 24 g/day. After delivery and through the lactation period, the mothers were fed ad libitum. The mothers were fed with a breeding diet (Euro Rodent Diet 22; 5LF5, Labdiet, Madrid, Spain) containing 55% carbohydrates, 22% protein, 4.4% fat, 4.1% fibre and 5.4% mineral at 12.2% humidity. Drinking water was provided ad libitum to all animals. Immediately after birth, the offspring were weighed individually and sexed, and the litter was randomly standardised to 12 rats, 6 males and 6 females, if possible. The rest of the litter was sacrificed with CO2. At the age of 6 months, male offspring from the two experimental groups (control and MUN) were analysed.

#### 2.1.2. Experimental Model of Spontaneous Hypertension

SHR, a well-known animal model of essential hypertension, was also used to make comparisons with the animal model of FPH induced by foetal undernutrition. Wistar-Kyoto rats (WKY) were chosen as the control model, representing a normotensive state. Both WKY and SHR male rats were bred at the Animal House of Universidad Autónoma de Madrid and used at the age of 6 months.

#### 2.1.3. Experimental Protocol

The animals from the different experimental groups (WKY, SHR, CONTROL and MUN) were first weighed and then anesthetized to measure the haemodynamic parameters (see below). Thereafter, the rats were sacrificed using a guillotine, the method being reported as the sacrificial advisable in studies involving the nervous system. The tissue samples were collected immediately after sacrifice and the mesenteric bed collected from which the main mesenteric artery was dissected. Four segments were obtained from each tissue.
