Prenatal cold exposure causes hypertension in offspring by hyperactivity of the sympathetic nervous system

Abstract

Environmental temperature plays a role in the variation of blood pressure. Maternal cold stress could affect the physiological phenotype of the offspring, including blood pressure elevation. In the present study, we found that adult offspring of dams exposed to cold have increased systolic and diastolic blood pressure, and decreased urine volume and sodium excretion, accompanied by increased heart rate and heart rate variability, secondary to increased activity of the sympathetic nervous system. Renal denervation or adrenergic receptor blockade decreased blood pressure and increased sodium excretion. The increase in peripheral sympathetic nerve activity can be ascribed to the central nervous system because administration of clonidine, a centrally acting α₂ adrenergic receptor agonist, lowered blood pressure to a greater degree in the prenatal cold-exposed than control offspring. Moreover, these prenatal cold-exposed offspring had hypothalamic paraventricular nucleus (PVN) disorder because magnetic resonance spectroscopy showed decreased N-acetylaspartate and increased choline and creatine ratios in the PVN. Additional studies found that prenatal cold exposure impaired the balance between inhibitory and excitatory neurons. This led to PVN overactivation that was related to enhanced PVN-angiotensin II type 1 (AT₁) receptor expression and function. Microinjection of the AT₁ receptor antagonist losartan in the PVN lowered blood pressure to a greater extent in prenatal cold-exposed that control offspring. The present study provides evidence for overactive peripheral and central sympathetic nervous systems in the pathogenesis of prenatal cold-induced hypertension. Central AT₁ receptor blockade in the PVN may be a key step for treatment of this type hypertension.

Keywords: Cold exposure; central AT1 receptor; hypertension; natriuresis; prenatal programing; sympathetic nervous system.

Figure 1.. Effect of prenatal cold stress on blood pressure, urine volume, sodium excretion, and heart rate in adult rat offspring

(A,B) Effect of prenatal cold stress on systolic (A) and diastolic (B) blood pressure in adult offspring. SBP and DBP were recorded by the tail-cuff method in conscious prenatal cold-exposed and control offspring at 4–24 weeks of age (*P<0.05 vs control, n=10/group). (C,D) Effect of prenatal cold stress on urine volume (C) and urine sodium (D) in adult offspring. Urine was collected in prenatal cold-exposed and control offspring at 4–24 weeks of age (*P<0.05 vs cold group, n=10/group). (E) Effect of prenatal cold stress on heart rate in adult offspring. The heart rate was measured at 4, 8, 12, 16, 20, and 24 weeks of age in conscious rats (*P<0.05 vs control, n=10/group).

Figure 2.. Effect of prenatal cold stress on HRV

Three HRV parameters, including LF/HF (A), SDNN (B), and RMSSD (C), were analyzed in prenatal cold-exposed and control offspring at 12 weeks of age to determine the SNS activity (*P<0.05 vs control, n=5/group).

Figure 3.. Effect of adrenergic receptor blockers, propranol and prazosin, on blood pressure and natriuresis

Effect of adrenergic receptor blockers, propranolol and prazosin, on SBP (A), DBP (B), urine volume (C), and urine sodium (D) in adult rat prenatal cold-exposed and control offspring. Propranolol (non-selective β antagonist, 30 mg/kg) and prazosin (specific α1 adrenergic receptor antagonist, 1 mg/kg) were administered into the stomach with a gastric tube twice daily for 1 week from 11 to 12 weeks of age. SBP and DBP were recorded by the tail-cuff method in conscious prenatal cold-exposed and control offspring. (*P<0.05 vs control, ^#P<0.05 vs others, n=5/group).

Figure 4.. Effect of renal denervation on blood pressure, urine volume, and urine sodium in adult rat prenatal cold-exposed and control offspring at 12 weeks of age

(A,B) Effect of renal denervation on blood pressure in adult rat prenatal cold-exposed offspring. SBP (A) and DBP (B) were recorded by the tail-cuff method in conscious prenatal cold-exposed and control offspring. (*P<0.05 vs control, ^#P<0.05 vs others, n=4/group). (C) Effect of renal denervation on urine volume in prenatal cold-exposed and control offspring (*P<0.05 vs control, #P<0.05 vs others, n=4/group). (D) Effect of renal denervation on urine sodium in adult prenatal cold-exposed and control offspring (*P<0.05 vs control, ^#P<0.05 vs others, n=4/group).

Figure 5.. Effect of centrally acting α2 adrenergic receptor agonist, clonidine, on blood pressure and natriuresis

Effect of centrally acting α2 adrenergic receptor agonist, clonidine, on SBP (A), diastolic blood pressure (B), urine volume (C), and urine sodium (D) in adult rat prenatal cold-exposed and control offspring at 12 weeks of age. Clonidine (0.2 mg/kg) was administrated into the stomach with a gastric tube twice daily for 1 week from 11 to 12 weeks of age. The blood pressure, urine volume, and urine sodium were measured after the last gavage. (*P<0.05 vs others, ^#P<0.05 vs control group without treatment, n=5/group).

Figure 6.. Effect of prenatal cold stress on the metabolic and structural changes of PVN neurons in adult rat offspring at 12 weeks of age

(A,B) MRS values of adult prenatal cold-exposed and control offspring. The ratios of NAA/Cr (A) and Cho/Cr (B) of the PVN region were acquired by the MRS to detect the central SNS disorder (*P<0.05 vs control, n=6). (C) The intensity of inhibitory and excitatory neurons changes in adult prenatal cold-exposed and control offspring. The excitatory glutamatergic neurons were identified by GRIN2B staining and the inhibitory GABAergic neurons were identified by GAD65 staining. The number of positive cells (black arrow) was counted in ten random fields of the PVN zone from each rat. (*P<0.05 vs control, scale bar = 100 μm, n=4/group).

Figure 7.. Effect of prenatal cold stress on AT₁ receptor expression and function in PVN of adult rat offspring at 12 weeks of age

(A,B) AT₁ receptor expression was detected by immunoblotting (A) and immunohistochemistry (B). (A) Results of immunoblotting are expressed as the ratio of AT₁ receptor and GAPDH (*P<0.05 vs control, n=6). (B) The staining in each group was repeated at least five times, and ten fields of vision in each slide were chosen for OD assessment (*P<0.05 vs control, scale bar = 100 μm, shown as black arrow). (C) Effect of the AT₁ receptor antagonist, losartan, on SBP in adult prenatal cold-exposed and control offspring. Losartan (AT₁ receptor antagonist) was administrated into the hypothalamic PVN by microinjection (11.45 nmol/kg). The rats were anesthetized with pentobarbital and blood pressure was measured from the femoral artery. Blood pressures were obtained 1 h after the losartan PVN injection (*P<0.05 vs others, ^#P<0.05 vs control group without treatment, n=8/group).

Figure 8.. Effect of losartan on the levels of inflammation and oxidase stress in PVN of prenatal cold exposed offspring at 12 weeks of age

(A–C) The levels of the pro-inflammatory cytokines IL-1β (A), IL-6 (B) and TNF-α (C) were measured by ELISA kits (*P<0.05 vs others, n=8). (D,E) The ROS level was measured by DHE staining and NADPH oxidase activity kit (*P<0.05 vs others, n=5). (F,G) MRS values of cold-exposed offspring with or without losartan. The ratios of NAA/Cr (F) and Cho/Cr (G) of the PVN region were acquired by the MRS to detect the central SNS disorder (*P<0.05 vs prenatal cold treated group, n=6).