Experiments were performed using Wistar rats in accordance with the European Convention on the protection of animals used for scientific purposes (EU Directive 2010/63/EU). Animal procedures were approved by the Moscow State University committee on animal welfare (protocol 133-g, approval date 08.10.2020). Sexually mature male and female Wistar rats were obtained from the Federal State Budgetary Scientific Institution of Research Institute of General Pathology and Pathophysiology Russian Academy of Sciences and then bred in the laboratory animal unit of the Biological Faculty, Moscow State University. The animals were maintained on 12 h light-dark cycle and had free access to the normal rodent chow (Laboratorkorm, Moscow, Russia).
4.1. Model of Fetal NO Deficiency in the Rat
Suppression of NO production in the prenatal and neonatal periods in rat pups was carried out by suppression of NO production in the organism of their mothers. For this purpose, sexually mature male and female rats were placed together overnight. The onset of pregnancy was determined by the presence of sperm in the vaginal smear the next morning (this day was considered the first GD). On GD10, females were randomly assigned to control or L-NAME groups. Females of the L-NAME group received L-NAME in drinking water (at a concentration of 500 mg/L) from GD10 until delivery, while females of the control group received tap water. During the pregnancy, the females of both groups were regularly weighed, and their water intake was recorded.
During the week before mating, female systolic BP and heart rate were recorded twice using tail-cuff plethysmography (Systola, Neurobotics, Russia). The first recording served to familiarize the animals with the procedure and the second one provided the pre-pregnancy values of hemodynamic parameters. The recordings were also performed on GD 5-7, GD15, and GD20. At every time point, the measurements were performed at least 5 times for each rat, the obtained values were averaged. On GD18-19, blood samples were taken from the incision of the tail tip (about 300 μL) from the females to determine the content of NO metabolites.
Rat pups of both sexes born to pregnant females were decapitated on postnatal days 1–2 (humane endpoint due to high incidence of limb defects). Trunk blood was collected, the heart and both kidneys were weighed and the aorta was isolated. For functional studies, two 2 mm long ring segments were cut from the thoracic aorta between the aortic arch and the diaphragm. Tissue samples collected for gene expression included the thoracic aorta and a proximal part of the abdominal aorta, these samples were placed in RNA-later (Qiagen) and stored at −20 °C until further analysis.
4.4. Functional Experiments on the Isolated Aorta
Aortic ring segments were mounted on steel wires in a multichannel myograph (620M, DMT, Denmark) for isometric force recording. The signal was digitized at a frequency of 10 Hz using an analog-to-digital converter (E14-140, L CARD, Russia); registration was carried out using the PowerGraph 3.3 software (DISoft, Russia). Throughout the experiment, solutions contained in myograph chambers were continuously aerated with carbogen (95% O2 + 5% CO2), to oxygenate and maintain pH 7.4.
After heating to 37 °C, the optimal passive stretch of the preparation was determined [34
] in a calcium-free solution (composition: NaCl—120 mM, NaHCO3
—26 mM, KCl—4.5 mM, MgSO4
—1 mM, NaH2
—1.2 mM, D-glucose—5.5 mM, EGTA—0.1 mM, HEPES—5 mM) in the presence of 1 μM NO donor DEA/NO. The preparations were stretched step by step and the force (F) corresponding to the given stretching of the preparation was recorded for 2 min at each step. From this value of the force, the passive tension
was calculated, where L is the length of the aortic segment. Using the Laplace equation, the transmural pressure
was calculated, where R is the inner radius of the vessel, IC is the internal circumference of the preparation corresponding to the radius R. The internal circumference of the preparation at each step of its stretching was calculated as
, where 40 µm is the diameter of the steel wires on which the preparation was mounted, and a is the distance between the wires. Using the obtained values, the relationship of the inner radius on pressure was plotted and approximated by the equation
, where P0
is the pressure corresponding to the adjacent position of the wires, and K is a constant. Using the constant K, the aortic diameter was calculated in the pressure range from 20 to 120 mm Hg. The inner diameter of the vessel corresponding to a pressure of 100 mm Hg (d100
) was calculated as well.
Further, the aortic rings were set to the optimal stretch corresponding to 0.9d100 and placed in a solution of the following composition: NaCl—120 mM, NaHCO3—26 mM, KCl—4.5 mM, MgSO4—1 mM, NaH2PO4—1.2 mM, D-glucose—5.5 mM, EDTA—0.025 mM, HEPES—5 mM, and 5–10 min later the cumulative addition of increasing concentrations of Ca2+ was carried out into the myograph chamber in the concentration range from 10 μM to 3 mM (the exposure to each concentration lasted for 3 min). To determine the maximum contractile response, the preparations were additionally stimulated with 60 mM KCl. After that, the preparations were washed with a solution of the following composition: NaCl—120 mM, NaHCO3—26 mM, KCl—4.5 mM, CaCl2—1.6 mM, MgSO4—1 mM, NaH2PO4—1.2 mM, D-glucose—5, 5 mM, EDTA—0.025 mM, HEPES—5 mM. This solution was used during the further experiment.
Relaxation responses of aortic segments were studied against the level of spontaneous precontraction which approximated the level of the maximal contractile response (Figure 2
a). Relaxation responses to acetylcholine and the role of NO in this response were studied using two adjacent segments of the same aorta. The NO synthase inhibitor L-NNA (100 μM) was added to one segment, and the same volume of solvent (H2
O, 50 μL) was added to the other. Twenty minutes later, basal tone values recorded in the absence and the presence of L-NNA were 88.5 ± 3.2% and 94.4 ± 1.1% in the control group and 81.8 ± 2.3% and 83.6 ± 2.1% in the L-NAME group (no statistically significant effects of either group or L-NNA were observed using two-way ANOVA with Sidak posthoc test). Then the concentration-response relationship to acetylcholine was carried out for both segments (concentration range 10 nM—10 μM, the duration of each concentration action was 2 min). After washing from acetylcholine, 100 μM L-NNA was re-added to the previously L-NNA-treated segment and 20 min later the concentration-response relationship on DEA/NO was carried out (concentration range 10 nM—10 μM, the duration of each concentration action was 2 min). Basal tone values before application of the first DEA-NO concentration were 69.5 ± 3.6% in the control group and 78.0 ± 2.9% in the L-NAME group (p
> 0.05 unpaired Student’s t
-test). At the end of the experiment, 2 mM EGTA was added to all preparations to determine the level of force that corresponded to the complete relaxation of smooth muscle (the level of “passive” force).
During data analysis, the value of the “passive” force was subtracted from all force values recorded during the experiment. The obtained values of active force were expressed as a percentage of the maximum force of contraction (for contractile responses to Ca2+) or a percentage of the precontraction level (for relaxation responses to acetylcholine or DEA/NO). For the reactions of the aorta to acetylcholine, the area above the relaxation curve was calculated using the GraphPad Prism 7.0 software (San Diego, CA, USA).