*2.5. Immunohistochemistry*

Immunohistochemistry procedures were previously described [53]. Briefly, four artery segments were obtained from each artery and immediately placed in cold phosphate buffer solution (PBS; in g/L): NaCl 8.0, Na2HPO4.2H2O 0.77, KCl 0.20 and KH2PO4 0.19 (pH 7.2). Each segment was longitudinally opened and fixed (paraformaldehyde 4% PBS; 50 min; room temperature). After two 15-min PBS washing cycles, artery segments were incubated with the primary antibody (mouse monoclonal anti-tyrosine hydroxylase, TH, 1:100 dilution, overnight, 4 ◦C to stain noradrenergic nerve terminals). Thereafter, tissues were incubated with Alexa 488 anti-mouse fluorescent secondary antibody (1:1000 dilution, 1 h, room temperature). The primary antibody was previously validated by the manufacturer. Negative controls were incubated on adjacent sections using 10% normal horse serum or blocking solution instead of the primary antibody. After three PBS washing cycles, the arteries were mounted intact with antifading agent (headshield mounting medium with DAPI).

Artery segments were visualized with a Leica SP2 laser scanning confocal microscopy (LSCM) system (Leica Microsystems, Metzler, Germany) fitted with an inverted microscope (×63 oil immersion lens). Stacks of 1-μm-thick serial optical images were captured from five randomly chosen regions along the adventitial layer of the mesenteric artery, which was identified by the shape and orientation of the nuclei stained with DAPI [55]. Adventitia was scanned along each mesenteric artery and the resulting images were reconstructed separately for each wavelength. Two stacks of images were sequentially obtained at different wavelengths: the first stack was taken with the Ex 405 nm and Me 412–470 nm wavelength to visualize cell nuclei (DAPI staining). The second was taken with the Ex 488 nm and Em 490–570 nm wavelength to visualize the TH (location of sympathetic terminals). Image acquisition was always performed under the same laser power, brightness, and contrast conditions. The resulting images were reconstructed separately for each wavelength for later quantification.

Quantitative analysis of confocal z-stacks images was performed using image analysis software (PAQI, CEMUP, Porto, Portugal) as previously described [56]. Briefly, a sequential routine was designed and developed to analyse each fluorescent signal used. PAQI software measured the surface area and strength of the fluorescence signal marking the postganglionic sympathetic nerves.

#### *2.6. Histology*

Serial 2-μm thickness sections of mesenteric arteries, previously fixed in paraformaldehyde 4% PBS, were dewaxed in xylene, then they were hydrated in decreasing concentrations of alcohols and stained with orcein, haematoxylin/eosin and with Masson Trichrome. Each tissue was cut in five levels along the length of the vessel to ensure data represents the putative mesenteric artery heterogeneity rather than only a specific location of the

artery. Each batch represents histochemical staining and includes sections from all five levels of mesenteric artery from each animal group. This procedure was repeated three times (3 batches). In total, 150 sections were obtained. Sections were stained and divided according to animal source. Within each of these groups, a random selection of the sections was carried out.

Stained sections were visualized using a high-resolution Zeiss Axiocam 105 colour digital camera mounted on a Zeiss Primo Star microscope, using an ×10 objective, to analyse the arterial lumen, media and adventitia layer. Histomorphometry was performed with ImageJ software [57], and data of lumen diameter and cross sectional area of the media and the adventitia were obtained.

#### *2.7. Statistics*

Statistics were performed with GraphPad Prism (version 8.3) software (San Diego, CA, USA). Sample size was calculated assuming a probability error of alpha type of 5% (*p* < 0.05) and potency of 80%. The normality of the variables was evaluated with Kolmogorov-Smirnov test. Results were expressed as mean ± s.e.m. Differences of means were compared using one- or two-way ANOVA, followed by post-hoc Holm-Sidak's multicomparison *t* test or Student's *t* test. A *p* value lower than 0.05 was considered to denote statistically significant differences.

#### **3. Results**

Body weight was not different between male MUN (460.18 ± 8.1 g; *n* = 6) and CON-TROL rats (486.6 ± 18.6 g; *n* = 6; *p* = 0.21). Moreover, no differences between body weight of SHR (377.4 ± 8.9; *n* = 6) and WKY (367.0 ± 11.9 g; *n* = 6, *p* = 0.52) were found.

MUN exhibited larger SPB and DBP, but not HR, when compared to CONTROL, whereas SHR evidenced larger SBP, DBP and HR when compared to WKY rats (Table 1). MUN showed lower SBP and DBP levels compared to SHR. However, HR was not enhanced in MUN, contrasting with the HR values observed in SHR.

**Table 1.** Blood Pressure and Heart Rate from CONTROL/MUN and WKY/SHR rats.


Four animal groups in the study: MUN, offspring from rats exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate. Values are mean ± s.e.m, from *n* rats from each group in study. Significant differences from the appropriate animal control: \* *p* < 0.05; from the WKY model: † *p* < 0.05; from the SHR model: # *p* < 0.05.

#### *3.1. Influence of Foetal Undernutrition on Vascular Morphology*

Histological sections of the mesenteric artery showed a reduced lumen area in MUN compared to CONTROL (Figures 1a and 2b), a reduction of MUN arterial lumen near 0.6-fold relatively to CONTROL values. Such effect was also observed in arteries from SHR and WKY, a reduction of SHR arterial lumen near 0.7-fold relatively to WKY values.

**Figure 1.** Histomorphometry of the lumen of mesenteric arteries from CONTROL and MUN (upper panel) and WKY and SHR rats (lower panel). (**a**) Images were obtained from orcein stained arteries (scale bar = 500 μm); (**b**) the graphics show the lumen area. MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. Values are mean ± s.e.m. from 6 rats of each group. Significant differences from the respective control rat: \* *p* < 0.05.

The media and adventitia layers of MUN were thicker compared to CONTROL (with an increase near 1.3-fold and 1.5-fold, respectively) similarly to what occurred in SHR compared to WKY (with an increase near 1.4-fold and 1.5-fold, respectively) (Figure 2a,b). Moreover, ratios of media/lumen and of adventitia/lumen were significantly increased in both MUN and SHR compared to data from respective controls, CONTROL and WKY (Table 2).


**Table 2.** Vascular wall morphology changes associated with hypertension.

Images were obtained from haematoxylin/eosin-stained arteries. Four animal groups in the study: MUN, offspring from rats exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. Values are mean ± s.e.m, from 6 rats from each group in study. Significant differences from the appropriate animal control: \* *p* < 0.05.

**Figure 2.** Histomorphometry of hypertensive (MUN and SHR) and normotensive (CONTROL and WKY) mesenteric wall. (**a**) Images were obtained from haematoxylin/eosin-stained arteries (scare bar = 300 μm). (**b**) Cross-sectional area of tunica media (right panel) and cross-sectional area of tunica adventitia (left panel). MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. Values are mean ± s.e.m. from 6 rats of each group. Significant differences from the respective control rat: \* *p* < 0.05; \*\* *p* < 0.01 \*\*\* *p* < 0.001.

An increase in the connective tissue content was also observed, both in MUN and SHR, compared to their respective controls (CONTROL and WKY, respectively, Figure 3); the connective tissue increased in MUN near 2.1-fold relatively to CONTROL, whereas in SHR, it increased 1.5-fold relatively to WKY).

**Figure 3.** Histomorphometry of hypertensive (MUN and SHR) and normotensive (CONTROL and WKY) mesenteric arteries, stained with Trichrome. MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. Values are mean ± s.e.m. from 6 rats of each group. Significant differences from the respective control rat: \* *p* < 0.05.

#### *3.2. Influence of Foetal Undernutrition in Sympathetic Postganglionic Nerves Activation*

Electrical field stimulation (5 Hz, 1 ms, 100 pulses, 50 mA) tritium outflow was higher in mesenteric arteries from MUN compared to CONTROL (Figure 4). Similarly, there was a larger tritium outflow in mesenteric arteries from SHR compared to WKY (Figure 4). The fractional rate of basal tritium outflow (b1), electrically evoked tritium overflow (S1) and S2/S1 ratios are shown in Table 3. Basal outflow and electrically evoked tritium overflow remained constant throughout the control experiments, with bn/b1 and Sn/S1 values close to unity. Electrically evoked tritium overflow (S1) was higher in hypertensive arteries (both SHR and MUN) compared to their respective control (WKY or CONTROL) vessels (Table 3). However, the evoked overflow was similar between the hypertensive arteries (MUN *versus* SHR arteries: *p* > 0.05).

**Figure 4.** Representative examples of time course tritium outflow from: mesenteric arteries from normotensive animals, CONTROL (circles, left panel) and WKY (circles, right panel), and hypertensive animals, MUN (triangles, left panel) and SHR (triangles, right panel) from typical experiments. After pre-incubation with [3H]-noradrenaline, tissues were superfused with [3H]-noradrenaline-free medium containing desipramine (400 nM). Tritium outflow (ordinates) is expressed as a percentage of the total radioactivity present in the tissue at the beginning of the collection period and was measured in samples collected every 5 min. Artery segments were stimulated twice by 100 pulses/5 Hz, (S1, S2). Each line represents the outflow of tritium from a single superfusion chamber; MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats.


**Table 3.** Basal tritium outflow (b1), electrically evoked tritium overflow (S1) and S2/S1 ratios from mesenteric arteries from CONTROL/MUN and WKY/SHR rats.

Tissues were stimulated twice at 30-min intervals (S1–S2; 100 pulses, 5 Hz, 1 ms, 50 mA): b1 refers to the 5-min period immediately before S1. The electrically evoked tritium overflow is expressed as a percentage of the tissue tritium content at the onset of stimulation. Basal tritium outflow (b1), electrically evoked tritium overflow (S1) from the four animal groups in study. MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. Values are mean ± s.e.m, from 6 rats from each group in study. Significant differences from the appropriate animal control: \* *p* < 0.05.

#### *3.3. Influence of Foetal Undernutrition on Perivascular Sympathetic Innervation*

In our experimental conditions, total tissue tritium content (per mg of tissue) was higher in MUN compared to CONTROL arteries (Figure 5a): total tissue content in MUN increased near 2.1-fold relatively to CONTROL. Moreover, tritium uptake was also larger in SHR mesenteric arteries compared to the values from WKY arteries (Figure 5b): total tissue content in SHR increased 1.7-fold relatively to WKY.

**Figure 5.** [ 3H]-Tritium uptake in mesenteric arteries from (**a**) CONTROL and MUN and (**b**) WKY and SHR rats. MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. Values are mean ± s.e.m. from 6 animals per group. Significant differences from the respective control rats: \* *p* < 0.05.

The influence of foetal undernutrition on perivascular sympathetic innervation in the adventitial layer of mesenteric arteries (identified from LSCM images by the shape and orientation of the nuclei and by exhibiting scattered fibres [56], was also assessed using a sympathetic neuronal marker, thyroxine hydroxylase (TH). Non-significant immunoreactivity was observed when the primary antibody was omitted (negative controls, data not shown). In mesenteric arteries from all experimental groups, immunoreactivity for the sympathetic neuronal marker TH evidenced the presence of sympathetic nervous fibres (Figure 6a, green marker). However, the pattern of TH immunoreactivity in arteries from SHR and from MUN exceeded those observed in WKY and CONTROL arteries, respectively (Figure 6b), revealing a denser and thicker sympathetic innervation: TH immunoreactivity, in MUN, increased near 1.9-fold relatively to CONTROL, whereas, in SHR, it increased 1.5-fold relatively to WKY.

**Figure 6.** (**a**) Laser scanning confocal microscopy representative images of the adventitia layer of mesenteric arteries from CONTROL and MUN (upper panel) WKY and SHR (lower panel) rats. Images show the immunofluorescence reactivity to TH (green) with DAPI-stained nuclei (blue). Scale bar: 25 μm. (**b**) Quantitative analysis of LSCM images. MUN, offspring exposed to maternal undernutrition during pregnancy; CONTROL, offspring from mothers fed ad libitum during pregnancy; SHR, spontaneously hypertensive rats; WKY, Wistar Kyoto rats. In the graphics, values are mean ± s.e.m. from 6 rats from each group. Significant differences from the respective control rats: \* *p* < 0.05.
