**4. Discussion**

The current study shows that foetal undernutrition induces an increase in the arterial sympathetic neurotransmission in rat adulthood (6-months old) which can justify, at least in part, the arterial inward hypertrophic remodelling observed and further highlighted the role of adventitia in the pathophysiology of foetal programming of hypertension. In addition, this study also shows that MUN rats share with SHR, a rat model of essential hypertension, these pathological aspects, which may contribute to the development of hypertension.

Foetal undernutrition was associated with the development of sympathetic hyperinnervation observed in the adventitial layer of the mesenteric artery. LSCM data revealed the presence of nerve fibres positive for TH (a sympathetic nervous fibre marker), spread through the adventitia reaching the medial layer, being thicker in MUN mesenteric arteries. Tyrosine hydroxylase (TH) is an enzyme localized inside sympathetic neurotransmitter storage vesicles [58] that is used as a peripheral sympathetic marker [43,58]. The higher TH immunoreactivity observed (induced by undernutrition during foetal life) indicates the occurrence of a sympathetic hyperinnervation in MUN mesenteric arteries and is in accordance with a previous report in FPH induced by prenatal hypoxia, in which a sympathetic hyperinnervation in tibial arteries was described [59]. A denser innervation and higher NA content was also observed in SHR mesenteric arteries, in accordance with previous studies [54,60]. The larger innervation was associated with higher noradrenaline (NA) content and release, assessed by functional studies using [3H]-NA, which can be taken up into vesicles in sympathetic nerve terminals by a specific NA transporter [61,62]. The total tissue tritium content can be considered an indicator of the sympathetic innervation density [54]. The larger release of tritium upon electrical stimulation can be a consequence of the hyperinnervation observed. However, it may also reflect changes in the presynaptic machinery and regulation of noradrenaline release. We have previous evidence that these mechanisms are altered in both SHR [43,53,54] and MUN rats [63]. Our data also showed that the total tissue content of the MUN mesenteric artery was higher compared to CONTROL, indicating that the MUN nervous terminals can incorporate more NA. Our functional data showed a higher sympathetic output in both SHR and MUN rats, compared to their respective controls. While the difference in innervation was similar for both hypertensive models, the tritium outflow and NA content was relatively higher in MUN rats, suggesting an increased availability of NA in the synaptic cleft of sympathetic MUN nerves. Similar data was found in SHR mesenteric [54,64] and tail [65] arteries. Our data agrees with studies in other models of FPH showing that SNS activation seems to be increased [36,58,66–69]: it was described an increased circulating levels of NA in animal models of FPH [70–72] and in LBW humans [73].

An increased sympathetic neurotransmission can contribute to increase vascular tone and to the observed remodelling, which, in turn, can be one of the mechanisms behind hypertension development in MUN. When compared to SHR, the increased NA release observed was analogous to MUN, although SHR presented a smaller basal outflow. This data is in line with previous studies in SHR, in which a sympathetic hyperactivity was also described in the cerebral artery [74], tail artery [49] and mesenteric bed [52]. Morphometric analysis indicated that foetal undernutrition induces inward hypertrophic remodelling in the mesenteric artery, with the contribution of both media and adventitia layers, and the presence of vascular fibrosis, in agreement with data from other studies, in SHR [75] and MUN [76,77]. The MUN mesenteric artery exhibits a reduced lumen area as well as an increased media and adventitia layers compared to CONTROL, indicative of inward hypertrophic vascular remodelling. A similar profile of vascular wall changes between SHR and WKY was also observed. This type of remodelling is a characteristic of hypertension and can contribute to increase total peripheral vascular resistance [78]. In mesenteric arteries from rats exposed to undernutrition in utero, the same type of remodelling was also reported [79].

The hypertrophy observed in the media layer of MUN mesenteric artery can be due to cellular hypertrophy caused by vascular smooth muscle cell (VSMC) proliferation or growth. Sympathetic hyperinnervation was shown to be related to media hypertrophy in jejunal arteries of SHR, since an increase in the number of nerve fibres occurred before the development of hypertension or an increase in the thickness of the arterial media [79]. Such finding suggests that increased sympathetic activity possibly plays a causal role in the development of hypertension, through vascular remodelling. Our study proves that the remodelling process is relatively similar in MUN and SHR (inward hypertrophic).

MUN had higher SBP and DBP than CONTROL, confirming previous results with the same foetal model [11,45,80–82]. SHR also exhibited higher values of SBP compared to WKY, as shown in many studies [54,83–85]. It was not possible to compare the blood pressure directly between SHR and MUN male rats, since they have a control strain with different background. However, the current study indicates that foetal undernutrition induces a milder form of hypertension when compared to a genetic model of essential hypertension, with lower levels of blood pressure (around 50 mmHg difference for SHR-WKY and 25 mm Hg for MUN-SD control). Additionally, MUN rats did not show an elevated HR, which was detected in the SHR model. The lack of HR alterations of MUN rats is in line with other studies using maternal low protein [35,86] or low micronutrients, such as zinc [87] or vitamin B12 [88] diets during pregnancy. In SHR, the tachycardia probably reflects an alteration in baroreceptor regulation, as previously reported [86], which may contribute to the higher blood pressure in this strain. It should be considered that the blood pressure data was obtained under ketamine/medetomidine anaesthesia, which exerts an influence on the sympathetic nervous system: ketamine has been shown to reduce both exocytosis and NA uptake [89]; medetomidine has been reported to reduce noradrenaline outflow within the central nervous system, dampening the central sympathetic tone [90]. Given our findings of an increased sympathetic activity and innervation in both SHR and MUN rat, it is likely

that the blood pressure in both models of hypertension was underestimated. Accordingly, previous studies in adult SHR assessed by tail cuff showed high blood pressure levels over 220 mmHg of SBP in awake animals [91], relatively to those reported in the present study under anaesthesia (SBP = 186 mmHg). In MUN rats, recent reports in awake 6-month-old rats showed SBP values of 163.1 mmHg [92], which are higher than those reported in the present study under anaesthesia (SBP = 151.1 mmHg).

In SHR rats, sex differences have been associated, among other causes, with increased sympathetic outflow due to dysfunctional regulation of presynaptic α-adrenoceptors in males [93]. Regarding the MUN model, we have reported that females counteract better the effects of foetal stress and do not develop hypertension in adult life [46]. It is important to know that these studies were performed under medetomidine/ketamine anaesthesia. However, recent reports have revealed that, in non-anesthetized rats, there is a tendency toward hypertension in 6-month-old MUN female rats, which is established and evident in old age [91]. Such data suggested that MUN females may also have increased blood pressure levels, and this could also explain the similarities in vascular remodelling between males and females [81]. Thus, the analysis of sex differences in MUN models deserves further attention.

#### **5. Conclusions**

In conclusion, and as depicted in Figure 7, elevated sympathetic neurotransmission in MUN and SHR supports that SNS plays an important role in the development of hypertension in both foetal programming and essential hypertension, which is in line with the neurogenic hypothesis of hypertension [94]. The similarities regarding sympathetic neurotransmission and remodelling in both models suggest that the increased sympathetic activity observed in FPH plays a causal role in the development of hypertension through vascular remodelling.

**Figure 7.** Effect of suboptimal foetal undernutrition on SNS and vascular wall from a rat FPH model.

**Author Contributions:** Conceptualization, S.M.A. and C.D.; methodology, P.R.-R., J.B.S. and M.S.V.- R.; software, M.S.V.-R., S.M.A., J.B.S. and C.D.; validation M.S.V.-R., S.M.A. and C.D.; formal analysis, M.S.V.-R., S.M.A., J.B.S. and C.D.; investigation, M.S.V.-R., S.M.A. and C.D.; resources, M.S.V.-R., S.M.A. and C.D.; data curation, M.S.V.-R., S.M.A. and C.D.; writing—original draft preparation, M.S.V.-R., S.M.A. and C.D.; writing—review and editing, all authors; visualization, M.S.V.-R.; supervision, S.M.A. and C.D.; project administration, S.M.A. and C.D.; funding acquisition, S.M.A. and C.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** Portuguese Foundation for Science and Technology (FCT) is acknowledged for UIDB/QUI/ 50006/2020 and Spanish Ministry of Science and Innovation, COCARDIOLAC project (RTI 2018- 097504-B-I00).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Review Board of Universidad Autónoma de Madrid (CEI-UAM 96-1776-A286), the Regional Environment Committee of the Comunidad Autónoma de Madrid (PROEX 04/19; date of approval 5 July 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the author (M.S.V.-R.).

**Acknowledgments:** We would like to thank the personnel of the Animal Facilities at Universidad Autónoma de Madrid for the care of the animals.

**Conflicts of Interest:** The authors declare no conflict of interest.
