Intrauterine L-NAME Exposure Weakens the Development of Sympathetic Innervation and Induces the Remodeling of Arterial Vessels in Two-Week-Old Rats

Abstract

Nitric oxide (NO) has been shown to stimulate differentiation and increase the survival of ganglionic sympathetic neurons. The proportion of neuronal NOS-immunoreactive sympathetic preganglionic neurons is particularly high in newborn rats and decreases with maturation. However, the role of NO in the development of vascular sympathetic innervation has never been studied before. We tested the hypothesis that intrauterine NO deficiency weakened the development of vascular sympathetic innervation and thereby changed the contractility of peripheral arteries and blood pressure level in two-week-old offspring. Pregnant rats consumed NOS inhibitor L-NAME (250 mg/L in drinking water) from gestational day 10 until delivery. Pups in the L-NAME group had a reduced body weight and blood level of NO metabolites at 1-2 postnatal days. Saphenous arteries from two-week-old L-NAME offspring demonstrated a lower density of sympathetic innervation, a smaller inner diameter, reduced maximal active force and decreased α-actin/β-actin mRNA expression ratio compared to the controls. Importantly, pups in the L-NAME group exhibited decreased blood pressure levels before, but not after, ganglionic blockade with chlorisondamine. In conclusion, intrauterine L-NAME exposure is followed by the impaired development of the sympathetic nervous system in early postnatal life, which is accompanied by the structural and functional remodeling of arterial blood vessels.

Keywords: blood pressure; early postnatal development; nitric oxide; sympathetic innervation; vasculature.

Figure 1

Characteristics of pregnant female rats in the control (CON) and L-NAME groups. (a) Body weight gain during the pregnancy. (b) Water consumption by dams in the control and L-NAME groups. (c–d) Systolic blood pressure (BP, c) and heart rate (d) during the pregnancy, before (GD7), and during L-NAME treatment (GD15 and GD20); GD, gestation day; n, number of females. Data are presented as the mean ± S.E.M. * p < 0.05 compared to the control on the same day of pregnancy (unpaired Student’s t-test).

Figure 2

Body weights of the offspring of control (CON) and L-NAME-treated females at the 2nd (PND2) and the 14th postnatal days (PND14). Data are presented as the mean ± S.E.M., n, number of litters. * p < 0.05 (unpaired Student’s t-test).

Figure 3

The density of sympathetic innervation of the saphenous artery is reduced in two-week-old L-NAME offspring. (a–d) Representative images of the glyoxylic-acid-stained plexus of adrenergic nerve fibers in the saphenous artery walls of control (a,c) and L-NAME (b,d) offspring (scale bar: 50 μm). (e) The total length of adrenergic fibers in the saphenous artery of the offspring from the control and L-NAME groups. n, number of animals. Data are presented as the mean ± S.E.M., * p < 0.05 (unpaired Student’s t-test).

Figure 4

Concentration-dependent contractile response of saphenous arteries of two-week-old offspring from the control (CON) and L-NAME groups to the α₁-adrenoceptor agonist methoxamine. Data are presented as the mean ± S.E.M., n, number of animals. # p < 0.05 compared to respective value in the control group (two-way ANOVA followed by Šidák’s post hoc analysis).

Figure 5

Relative mRNA levels of α-actin (a), β-actin (b), α-actin/β-actin ratio (c), smooth muscle myosin heavy chain isoform (SM-MHC) (d), non-muscle myosin heavy chain isoform (NM-MHC) (e), SM-MHC/NM-MHC ratio (f) and α_1a-adrenoceptor (g) in the saphenous artery of control (CON) or L-NAME offspring. Data are presented as the mean ± S.E.M., n, number of animals. * p < 0.05 (unpaired Student’s t-test). Data are normalized to the mRNA content of RPLP0 in the same tissue sample; the mean value in the control group was taken as 100%.

Figure 6

Systemic cardiovascular parameters of anesthetized two-week-old offspring before and after the ganglionic blockade. Mean arterial pressure (a) and heart rate (b) values before (baseline) and after ganglionic blockade (chlorisondamine, 2.5 mg/kg) in pups from control (CON) and L-NAME groups. Data are presented as the mean ± S.E.M., n, number of animals. * p < 0.05 (unpaired Student’s t-test).