Developmental Maturation and Alpha-1 Adrenergic Receptors-Mediated Gene Expression Changes in Ovine Middle Cerebral Arteries

The Alpha Adrenergic Signaling Pathway is one of the chief regulators of cerebrovascular tone and cerebral blood flow (CBF), mediating its effects in the arteries through alpha1-adrenergic receptors (Alpha1AR). In the ovine middle cerebral artery (MCA), with development from a fetus to an adult, others and we have shown that Alpha1AR play a key role in contractile responses, vascular development, remodeling, and angiogenesis. Importantly, Alpha1AR play a significant role in CBF autoregulation, which is incompletely developed in a premature fetus as compared to a near-term fetus. However, the mechanistic pathways are not completely known. Thus, we tested the hypothesis that as a function of maturation and in response to Alpha1AR stimulation there is a differential gene expression in the ovine MCA. We conducted microarray analysis on transcripts from MCAs of premature fetuses (96-day), near-term fetuses (145-day), newborn lambs, and non-pregnant adult sheep (2-year) following stimulation of Alpha1AR with phenylephrine (a specific agonist). We observed several genes which belonged to pro-inflammatory and vascular development/angiogenesis pathway significantly altered in all of the four age groups. We also observed age-specific changes in gene expression–mediated by Alpha1AR stimulation in the different developmental age groups. These findings imply complex regulatory mechanisms of cerebrovascular development. For these studies, we used cerebral arteries from premature fetuses (gestational age - 96-days old), near-term fetuses (gestational age 140 days), newborn lambs (5 days old) and non-pregnant adult sheep (18-24 months) obtained from Nebeker Ranch (Lancaster, CA). For each experiment, four animals were used; in case of fetus twins only one of the twins was included in the study. In our previous studies, we have described the isolation of MCA from sheep 38,52,53. Briefly, pregnant and non-pregnant ewes were anesthetized with thiopental sodium (10mg/kg, Intravenous) and anesthesia was maintained with inhalation of 1% of isoflurane in oxygen throughout surgery. Following the delivery of the fetus by hysterotomy, fetuses and ewes were euthanized with an overdose of proprietary euthanasia solution, Euthasol (pentobarbital sodium 100mg/kg and phenytoin sodium 10mg/kg; Virbac, Ft. Worth, TX). Studies were performed in isolated MCAs cleaned of adipose and connective tissue. From each animal, cleaned MCA were cut into segments ∼4 mm length, and placed in tissue baths containing Krebs buffer (containing in mM: 120 NaCl; 4.8 KCl; 1.2 K2HPO4; 25 NaHCO3; 1.2 MgCl2; 2.5 CaCl2 and 10 glucose) bubbled with 95% O2/5% CO2 for 30 minutes. We exposed the arteries to PHE (10µM) at 38°C, the contraction of the arteries was monitored and following exposure for 30 minutes arterial segments were snap frozen in the organ bath and stored in -80 degrees for RNA isolation.