Brachiocephalic Occlusion Research Articles

Transcriptomics of the fetal hypothalamic response to brachiocephalic occlusion and estradiol treatment.

Estradiol (E2) is a well-known modulator of fetal neuroendocrine activity and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic artery occlusion (BCO) or sham occlusion. Half of the fetuses received subcutaneous pellets that increased plasma E2 concentrations within the physiological range. Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Analysis of the data by ANOVA revealed that E2 differentially regulated (DR) 561 genes, and BCO DR 894 genes compared with control and E2+BCO DR 1,153 genes compared with BCO alone (all P < 0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis but did not significantly involve genes known to directly respond to the estrogen receptor. Brachiocephalic occlusion upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain and downregulated neuropeptide synthesis. E2 upregulated immune- and apoptosis-related pathways after BCO and reduced kinase and epigenetic pathway responses to the BCO. Responses to BCO are different from responses to hypoxic hypoxia suggesting that mechanisms of responses to these two forms of brain hypoxia are distinct. We conclude that cerebral ischemia caused by BCO might stimulate lymphocyte infiltration into the brain and that this response appears to be modified by estradiol.

Modeling the transcriptomics response of the late‐gestation fetal hypothalamus to estradiol and brachiocephalic occlusion (910.10)

Estradiol (E2) is a well‐known modulator of fetal neuroendocrine activity, and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic occlusion or sham occlusion. Half of the fetuses received subcutaneous pellets that released E2 (~250 μg/day, increasing plasma E2 from 62±6 to 102±12 pg/mL). Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Data were analyzed by ANOVA, using JMP Genomics (SAS Institute), revealing that E2 differentially regulated (DR) 561 genes, BCO DR 894 genes and estradiol+BCO DR 1153 genes compared to BCO alone (all p&lt;0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis, but did not significantly involve genes known to directly respond to the estrogen receptor (contain ERE sequences). BCO upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain, and downregulated neuropeptide synthesis. E2 upregulated immune‐ and apopotosis‐ related pathways after BCO, and reduced kinase and epigenetic pathway responses to the BCO. Array results are consistent with the results of more limited hypotheses previously published from these experiments and real‐time PCR validation of neuropeptide and immune‐related genes (CRH, CRHBP, POMC, AGRP, IL1β, TNFα) confirmed the array results. We conclude that increased E2 plasma concentrations invoke epigenetic pathways and appear to exacerbate brain inflammation after BCO. Actions of E2 in the fetal brain might not be dependent on ERα‐mediated gene transcription.Grant Funding Source: Supported by HD033053 and DK076541

Chemoreflex Activity Increases Prostaglandin Endoperoxide Synthase mRNA Expression in the Late-Gestation Fetal Sheep Brain

Fetal sheep defend blood pressure, blood volume, and blood gases using baro- and chemoreflexes that influence autonomic and neuroendocrine responses. The local generation of prostanoids within the fetal brain is also an important component in activating hormone responses to these stimuli, but the relationship between the reflexes and prostanoid biosynthesis is unclear. The present study was performed to test the hypothesis that the abundances of prostaglandin biosynthetic enzymes in the fetal brain are dependent upon the activity of the baro- and chemoreflex pathways. We subjected chronically catheterized fetal sheep in late gestation to a 10-minute period of brachiocephalic occlusion (BCO), a stimulus that provokes brisk cardiovascular and neuroendocrine responses. We compared the central nervous system abundance of prostaglandin endoperoxide synthases 1 and 2 (PGHS-1 and PGHS-2) after BCO to (1) fetal sheep that had been subjected to BCO after chronic sinoaortic denervation plus bilateral vagotomy and (2) fetal sheep in which the N-methyl d-aspartate (NMDA) receptor antagonist, ketamine, had been administered prior to BCO. Abundances of messenger RNA (mRNA) for PGHS-1 and of mRNA and protein for PGHS-2 in fetal hippocampus were reduced significantly by either prior denervation or ketamine administration. Prostaglandin endoperoxide synthases 1 and 2 mRNA in pituitary were decreased and increased, respectively, by ketamine pretreatment. The results of this study are consistent with the conclusion that the expression of PGHS-1 and -2 in fetal hippocampus and pituitary are influenced by the baro- and/or chemoreflex pathways within the fetal brain in late gestation.

Influence of Estradiol and Fetal Stress on Luteinizing Hormone, Follicle-Stimulating Hormone, and Prolactin in Late-Gestation Fetal Sheep

Background: Hypotension and reduced cerebral blood flow secondary to brachiocephalic occlusion (BCO) stimulate various homeostatic physiological and endocrine responses. Our previous studies have also suggested a role of estradiol in augmenting the fetal stress response to BCO. Objectives: We tested the hypothesis that gonadotropins and/or prolactin (PRL) are upregulated in fetal pituitary in response to fetal stress and play a role in the response to BCO-induced stress. Methods: We performed 3 studies: one in which we measured ovine fetal pituitary PRL, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) mRNA throughout the latter half of gestation in order to better understand the ontogenetic changes upon which dynamic responses are superimposed; one in which we measured these mRNA abundances in response to BCO and/or estrogen treatment, and one in which we measured plasma LH responses to BCO in chronically catheterized late-gestation fetal sheep. Results: PRL gene expression is increased dramatically in the last 20% of gestation. LH and FSH mRNAs were unchanged except for a transient dip in the expression of LH in the last few days before the normal time of spontaneous parturition. Chronic treatment with estradiol decreased LH and FSH mRNA, but increased PRL mRNA abundance after BCO. In contrast, BCO alone increases the abundance of LH, but not FSH or PRL mRNA in fetal pituitary. Plasma LH concentrations were not increased in response to BCO. Conclusions: We conclude that the late-gestation fetal sheep responds to hypotensive stress with increases in LH mRNA but not LH secretion. LH, FSH and PRL changes are therefore unlikely to contribute to the fetal response to cerebral hypoperfusion.

Blockade of PGHS-2 inhibits the hypothalamus-pituitary-adrenal axis response to cerebral hypoperfusion in the sheep fetus

Decreases in fetal blood pressure stimulate homeostatic stress responses that help return blood pressure to normal levels. Fetal hypothalamus-pituitary-adrenal (HPA) axis responses to hypotension are mediated by chemoreceptor and baroreceptor reflexes and ischemia of the fetal central nervous system. Indomethacin, a nonselective inhibitor of prostaglandin endoperoxide synthase (PGHS)-1 and -2, attenuates the HPA response to hypotension in the fetus. The present study was designed to test the hypothesis that selective inhibition of PGHS-2 also inhibits the HPA response to cerebral hypoperfusion. We studied 13 chronically catheterized fetal sheep (126-136 days gestation). Five fetal sheep were subjected to intracerebroventricular infusion of nimesulide (0.01 mg/day), a specific inhibitor of PGHS-2, and eight were treated with vehicle (DMSO in water) for 5 days. Each fetus was subjected to a 10-min period of brachiocephalic occlusion, which decreased carotid arterial pressure approximately 75% and reflexively increased fetal plasma concentrations of ACTH, POMC, cortisol, and femoral arterial pressure, and decreased fetal heart rate. Nimesulide significantly inhibited the ACTH response to the BCO, while significantly augmenting the reflex cardiovascular response and altering fetal heart rate variability consistent with increased sympathetic nervous system activity. The results of this study demonstrate that the activity of PGHS-2 in the brain is a necessary component of the fetal HPA response to cerebral hypoperfusion in the late-gestation fetal sheep. These results are consistent with those of recent study, in which we demonstrated that the preparturient increase in fetal ACTH secretion depends upon PGHS-2 activity within the fetal brain.

Subclavian steal and rest pain in a case of brachiocephalic artery occlusion

A patient with multiple vessel disease presented with symptoms of significant bilateral upper extremity pain and weakness that was more significant on the right side. On carotid duplex scanning, brachiocephalic artery occlusion with retrograde flow was noted through the right common carotid and right vertebral arteries at rest. Furthermore, 50% to 90% occlusion of the left internal carotid was noted. Filling of the right subclavian artery was noted to be through the right vertebral and right common carotid arteries. Unlike isolated subclavian steal syndrome, brachiocephalic artery occlusion induces significant hemodynamic alterations in extracranial arterial flow, which normally produces no symptoms at rest, but may produce symptoms with exercise. The occurrence of subclavian steal phenomenon in the presence of brachiocephalic occlusion is extremely rare. The present case is the first to report a patient presenting with bilateral upper extremity rest pain in the presence of brachiocephalic artery occlusive disease.

Cerebral Hypoperfusion Increases Neuronal Nitric Oxide Synthase Abundance in the Ovine Fetal Brainstem

Nitric oxide (NO) is potent vasorelaxant in vascular smooth muscle and serves as a neurotransmitter in the brainstem, influencing the neural pathways that control cardiovascular function. Hypotension, and the resultant cerebral hypoperfusion, stimulates a series of cellular responses that modify the reflex responses to subsequent periods of hypotension. This study was performed to test the influence of brachiocephalic occlusion (BCO) on the abundance of nNOS in the fetal brainstem. Fifteen chronically catheterized, late gestation fetal sheep were subjected to a 10 minute period of cerebral hypoperfusion followed by reperfusion. Fetuses were euthanized 0, 0.5, 3, 8, and 18 hours after the onset of BCO (n=3 at each timepoint). Brain regions were rapidly dissected, snap‐frozen, and later processed for isolation of protein for Western Blot analysis. We tested for an upregulation in fetal brainstem nNOS abundance in response to brachiocephalic occlusion using anti‐nNOS antiserum (Transduction Labs) and anti‐β‐actin (Sigma) for normalization of protein loading. After normalization with β‐actin, nNOS protein abundance appeared to increase 3 hours after the onset of a 10‐min period of BCO.When analyzed by one‐way ANOVA, the apparent increase was not statistically significant (p=0.079). Analysis of a smaller data set, in which the 18 hour values were excluded yielded a statistically significant change (p=0.022). Analysis of the individual means by Bonferroni test revealed that the nNOS abundance was greater at 3 hours than at earlier or later times. We conclude that nNOS abundance is increased in fetal brainstem after a period of cerebral hypoperfusion, and we speculate that increased nNOS activity increases brainstem NO responses to subsequent periods of hypotension.

Cerebral Hypoperfusion Increases Estrogen Receptor Abundance in the Ovine Fetal Brain and Pituitary

Background/Aims: Estrogen is an important component of fetal neuroendocrine function in late-gestation fetal sheep; however, little is known about the regulation of estrogen receptor abundance in the brain and pituitary of fetuses. The present study was performed to test the hypotheses that estrogen receptor abundance in the fetal brain and pituitary are influenced by circulating estradiol concentrations and that they are acutely regulated after cerebral hypoperfusion. Methods: We studied 16 time-dated fetal sheep (124–128 days gestation) that were chronically catheterized and instrumented at least 5 days before study. Four groups (n = 4 each) were studied in which fetuses received estradiol (0.25 mg/day, producing physiological increases in fetal plasma estradiol concentrations) or placebo implants, and in which fetuses received a 10-min period of brachiocephalic occlusion (BCO) or sham-BCO. One hour after BCO or sham-BCO, fetuses were euthanized and tissues rapidly removed for analysis of estrogen receptors (ER)-α and -β at the mRNA and protein levels. Results: Both BCO and estradiol treatment were effective in changing ER expression, although the effects were region-specific. BCO dramatically increased ER-α in the pituitary and both ER-α and ER-β in the brainstem, while decreasing ER-α expression in the hypothalamus. Estradiol treatment decreased ER-α expression in the hypothalamus, whereas it increased ER-α expression in the brainstem, cerebral cortex and hippocampus. Conclusions: We conclude that the expression of ER-α and ER-β in the brain and pituitary of fetal sheep are influenced by circulating estrogen concentrations and acutely regulated in response to cerebral hypoperfusion.

Ketamine inhibits fetal ACTH responses to cerebral hypoperfusion

The present study tested the effect of ketamine on the fetal reflex responses of late-gestation sheep to brachiocephalic occlusion (BCO), a stimulus that mimics the reduction in cerebral blood flow that results from severe fetal hypotension. Ketamine, a dissociative anesthetic and known noncompetitive antagonist of N-methyl D-aspartate (NMDA) receptors, has previously been shown to impair chemoreceptor responsiveness. Studies from this laboratory suggest that fetal reflex ACTH responses to hypotension are largely mediated by chemoreceptors; therefore, we hypothesized that ketamine would inhibit the reflex hormonal response to BCO. Chronically catheterized fetal sheep were subjected to acute cerebral hypoperfusion through occlusion of the brachiocephalic artery. Fetal blood pressure and heart rate were continuously recorded, and fetal blood samples drawn during the experiment were analyzed with specific hormone assays. Our results demonstrate that ketamine attenuates hemodynamic responses to cerebral hypoperfusion and is a potent inhibitor of ACTH and proopiomelanocortin (POMC)/pro-ACTH release. These data support the hypothesis that fetal reflex responses hypotension are chemoreceptor mediated. Given the potency with which ketamine inhibits ACTH response to fetal hypotension, we suggest that the use of ketamine or other anesthetic or analgesic drugs that block or otherwise interact with the NMDA-glutamate pathways, in late pregnancy or in preterm newborns be reconsidered.

Expression of Nitric Oxide Synthase Isoforms in the Ovine Fetal Brain: Alteration by Hormonal and Hemodynamic Stimuli

Nitric oxide (NO) is synthesized in the brain through the action of three isoforms of nitric oxide synthase (NOS). The local generation of NO in neurons, glia, and vasculature modulates neuronal activity, as well as regional cerebral blood flow. We propose that, in the fetal brain, cerebral hypoperfusion alters the expression of NOS isoforms, and that estrogen administration modulates the NOS response to hypoperfusion. Sixteen chronically catheterized fetal sheep of known gestational age (124 to 128 days' gestation) were subjected to a 10-minute period of brachiocephalic occlusion (BCO) or to sham BCO; half of these fetuses were subjected to subcutaneous implant, which released 17beta-estradiol (E2; 0.25 mg/d) or placebo. Brain tissue was collected for mRNA and protein extraction 1 hour after the start of the BCO or sham BCO. All three isoforms of NOS were identified in fetal brain at both the mRNA and protein levels. BCO increased NOS1 (hippocampus, brainstem), NOS2 (hypothalamus), and NOS3 (hippocampus, cortex) at the protein level. Estradiol alone increased NOS1 (brainstem, cortex), NOS2 (hippocampus, hypothalamus), and NOS3 (brainstem, cerebellum) at the protein level, changes that were not mirrored at the mRNA level. The combination of BCO and estradiol produced smaller changes in NOS1 (brainstem, cortex), NOS2 (hippocampus, hypothalamus), and NOS3 (brainstem) protein levels than those produced by either stimulus alone. We conclude that the fetal brain expresses all isoforms of NOS, and that NOS expression is altered by both BCO and estradiol, but that the most prevalent effect of estradiol is to reduce specific NOS responses to cerebral hypoperfusion. The present results suggest the possibility that the neuroendocrine responses to estradiol and BCO are modulated by central nervous system (CNS) NO biosynthesis.

Differential Modulation of Ovine Fetal ACTH Secretion by PGHS-1 and PGHS-2

Background/Aims: We have previously demonstrated that prostaglandin generation within the fetal brain augments or partially mediates fetal reflex responsiveness to hypotension. The present study was performed to test the relative roles of prostaglandin endoperoxide synthases-1 and -2 (PGHS-1 and 2, or COX-1 and 2) as potential mediators of this interaction. Methods: Chronically catheterized and instrumented fetal sheep were subjected to transient brachiocephalic occlusion (BCO) after intracerebroventricular injection of resveratrol (PGHS-1 or COX-1 inhibitor), nimesulide (PGHS-2 or COX-2 inhibitor), or vehicle. Results: BCO decreased arterial pressure perfusing the fetal brain and stimulated increases in systemic blood pressure and heart rate as well as in circulating concentrations of ACTH. Inhibition of PGHS-1 and PGHS-2 had differential effects on fetal ACTH secretion. Pre-BCO concentrations of plasma ACTH increased in response to nimesulide, while the fetal ACTH response to BCO was delayed by resveratrol. Prior to the BCO, nimesulide also increased fetal blood pressure and decreased fetal heart rate. The injections of resveratrol and nimesulide did not alter placental biosynthesis of prostaglandins and therefore acted within the fetal brain. Conclusion: We conclude that prostaglandin generated in the fetal brain by the action of PGHS-1 augments fetal ACTH reflex responses to BCO but that, in contrast, the action of PGHS-2 is inhibitory to ACTH secretion.

Ketamine inhibits fetal reflex response to cerebral hypoperfusion

Fetal sheep respond to hypoxia, hypotension and hypovolemia with reflex increases in circulating concentrations of adrenocorticotropin (ACTH) and cortisol. Previous studies indicate that these reflex responses may be more dependent on afferent activity of arterial chemoreceptors than baroreceptors. This suggests that these responses will be vulnerable to drugs that inhibit the fetal chemoreflex. Ketamine, a dissociative anesthetic that blocks the fetal reflex response to maternal ventilatory hypoxia, may act in this manner. We hypothesized that ketamine would block the reflex ACTH, pro-opiomelanocortin (POMC), and cortisol response to brachiocephalic occlusion (BCO), a stimulus that mimics the reduction in cerebral blood flow that results from severe fetal hypotension. Chronically catheterized fetal sheep (125–135 days gestation) were subjected to 10 minutes of cerebral hypoperfusion induced by occlusion of the brachiocephalic artery with or without pretreatment with iv ketamine (3mg/kg). Fetal blood samples were assayed for plasma ACTH, POMC, and cortisol. BCO stimulated robust increases in plasma ACTH and POMC levels that were significantly inhibited in ketamine-treated fetuses. Plasma cortisol was also significantly increased by occlusion; however, this rise was not attenuated by ketamine. We conclude that ketamine does block fetal reflex ACTH and POMC but not cortisol response to BCO. These data agree with our previous results demonstrating that sinoaortic denervation of chemoreceptors and baroreceptors prior to occlusion attenuates fetal ACTH stimulation and suggests that ketamine may inhibit chemoreflex and/or baroreflex pathways. Supported by HD42135 and an AHA pre-doctoral fellowship.

Fetal brain regional responses to cerebral hypoperfusion: modulation by estrogen

We have previously demonstrated that cerebral hypoperfusion stimulates several physiological and molecular responses which are components of homeostatic reflexes. Physiological increases in fetal plasma estradiol concentration modulate fetal brain responsiveness to hypotension. In the present study, we tested the effect of cerebral hypoperfusion and/or estradiol on the expression of Fos, the protein product of the gene c- fos in late-gestation fetal sheep. We hypothesized that estrogen and cerebral hypoperfusion alone would augment Fos abundance in various brain regions, including the hypothalamus and brainstem, and that estrogen would augment or otherwise modify the Fos response to cerebral hypoperfusion. Singleton or twin fetuses of time-dated pregnant ewes were chronically catheterized and fitted with an extravascular balloon occluder around the brachiocephalic artery using aseptic techniques. In one-half of the fetuses, we implanted a pellet subcutaneously which released estradiol at a rate of 5 mg in 21 days. Fetuses were studied at least 5 days after surgery (124–128 days' gestation, term is approximately 147 days). One-half of the fetuses were subjected to a 10-min period of brachiocephalic occlusion (BCO). One hour after the start of the experiment, the ewe and fetus were euthanized and the fetal brain was rapidly recovered, dissected, and frozen in a polypropylene tube in an acetone/dry ice bath. Brain tissue was homogenized in a boiling lysis buffer, and protein concentrations measured using the Bradford method. Extracted proteins were electrophoresed on 7.5% polyacrylamide gels, transferred to nitrocellulose membranes, and probed for Fos. In most brain regions, estradiol or BCO altered the expression of Fos. Analyzed by two-way analysis of variance, there was a statistically significant ( p<0.05) interaction between estradiol and BCO in brainstem, cerebellum, and hippocampus, nearly significant in hypothalamus ( p=0.07) and not statistically significant in cerebral cortex. In these regions with statistically significant interactions, the expression of Fos in response to the combined treatment of estradiol and BCO was less than the sum of responses to either treatment alone. We conclude that estradiol has a potent action on the fetal brain which is identifiable in the brainstem, cerebellum, and hippocampus and that it modulates the Fos response to cerebral hypoperfusion. The measurement of regional Fos responses using Western blot reveals a negative interaction between estrogen and BCO which might result from alterations in cerebral blood flow or metabolism.

Central nervous system prostaglandin endoperoxide synthase-1 and -2 responses to oestradiol and cerebral hypoperfusion in late-gestation fetal sheep.

Previous work in this laboratory has demonstrated that cerebral hypoperfusion increases the expression of prostaglandin endoperoxide synthase-2 (PGHS-2) in ovine fetal brain regions. Endogenously produced prostaglandins, in turn, partially mediate the fetal hypothalamus- pituitary-adrenal (HPA) axis response to arterial hypotension. In separate experiments, we have found that oestradiol stimulates fetal HPA axis activity. The present experiments were designed to test the hypothesis that oestradiol increases the expression of PGHS isoforms, and that oestradiol augments the PGHS response to cerebral hypoperfusion. Sixteen fetal sheep of known gestational ages (124-128 days' gestation at the time of study) were subjected to chronic catheterization and implantation of extravascular occluder around the brachiocephalic artery. Eight fetuses were subjected to subcutaneous implantation of a pellet containing 17beta-oestradiol (release rate 5 mg (21 days)-1). Brachiocephalic occlusion (BCO) stimulated adrenocorticotropin (ACTH), cortisol and arginine vasopressin (AVP) secretion, responses that were augmented by oestradiol. One hour after the beginning of a 10 min period of BCO, PGHS-1 mRNA was increased in fetal brainstem and hypothalamus, and PGHS-2 mRNA was increased in fetal brainstem. Oestradiol, by itself, increased the abundance of PGHS-2 mRNA in brainstem and cerebellum, and augmented the PGHS-2 mRNA response to BCO in brainstem. In contrast, oestradiol had no significant effect on the abundance of PGHS-1 mRNA in any brain region. PGHS-1 and PGHS-2 protein levels did not reflect the changes in the respective mRNAs. The abundance of both proteins was increased in cerebral cortex in response to BCO, and the abundance of PGHS-2 protein was increased by both oestradiol and BCO in the hippocampus. The results of this study confirm and extend the results of our previous studies, demonstrating an effect of cerebral hypoperfusion on the expression of both isoforms of PGHS. We conclude that oestradiol increases the expression of PGHS-2 in specific fetal brain regions, and that there is an interaction between oestradiol and BCO in the control of PGHS-2 expression in the fetal brainstem. We expect that at later time-points, the changes in mRNA would be followed by similar changes in enzyme abundance at the protein level. We speculate that at least a part of the effect of oestradiol on fetal HPA axis function is mediated by an interaction between oestradiol and prostaglandin biosynthesis in the fetal brain.

Induction of immunoreactive prostaglandin H synthases 1 and 2 and fos in response to cerebral hypoperfusion in late-gestation fetal sheep

The goal of the present study was to localize and quantify immunoreactive prostaglandin H synthase 1 (PGHS-1), PGHS-2, and Fos expression by immunohistochemistry in the fetal brain 30 minutes and 2 hours after the onset of a 10-minute period of cerebral hypoperfusion in barodenervated and chemodenervated fetal sheep. Fetal sheep of known gestational age were studied intact or sinoaortic denervated. Fetuses were sacrificed and tissues recovered for immunohistochemistry or real-time polymerase chain reaction measurements of protein and mRNA, respectively. Some fetuses were subjected to brachiocephalic occlusion, produced by inflation of an extravascular balloon occluder around the brachiocephalic artery. Immunohistochemistry results were quantified using image analysis, and mRNA was quantified by estimation of cycle threshold in generation of PGHS-1 or PGHS-2 amplicons. Sinoaortic denervation by itself did not alter the abundance of PGHS-1 or PGHS-2 protein in any brain region, although the denervation did reduce the abundance of PGHS-1 mRNA in hypothalamus. We assessed PGHS-1, PGHS-2, and Fos immunoreactive protein abundance by image analysis of histologic sections stained for the respective proteins using immunohistochemistry. Cerebral hypoperfusion increased the intensity of staining of immunoreactive PGHS-1, PGHS-2, and Fos in the anterior pituitary, hippocampus, and cerebellum. In the cerebral microvasculature, the intensity of PGHS-1 and Fos was significantly greater, and in the cerebral cortex, the intensity of PGHS-2 was significantly greater. Changes in the amount of immunostaining in the nucleus of tractus solitarius and paraventricular nucleus were not statistically significant. Cerebral hypoperfusion altered the expression and distribution of prostaglandin biosynthetic enzymes in ovine fetal brain by a mechanism that is independent of baroreceptor and chemoreceptor afferent activity.

Induction of PGHS-2 mRNA in response to cerebral hypoperfusion in late-gestation fetal sheep

Previous studies have demonstrated that cerebral ischemia stimulated the increased abundance of immunoreactive PGHS-2, but not PGHS-1, in brain tissue homogenates in late-gestation fetal sheep. The goal of the present study was to detect PGHS-1 and PGHS-2 mRNA in specific fetal brain regions, and to semi-quantitatively detect changes in the abundance of the respective mRNA’s in response to cerebral hypoperfusion. Fetal brain tissues were collected from control fetuses and from fetuses 30 min and 2 h after cerebral hypoperfusion (produced by brachiocephalic occlusion). Messenger RNA was studied by RT-PCR, and expressed semiquantitatively as a ratio of PGHS-1 or PGHS-2 mRNA abundance to β-actin mRNA abundance. PGHS-2 mRNA was only detected in the fetal hippocampus, hypothalamus, and brain stem and it was induced by cerebral hypoperfusion. In contrast, PGHS-1 mRNA was detected in all fetal brain tissues but was not induced. We conclude that cerebral hypoperfusion induced PGHS-2 gene expression in hippocampus, hypothalamus, and brainstem, and we speculate that the increased abundance of the enzyme is likely to be important for control of reflex responses to hypotension in the fetus.

Cerebral circulation in sleep: vasodilatory response to cerebral hypotension.

Little is known of the factors that regulate CBF in sleep. We therefore studied 10 lambs to assess the vasodilatory processes that underlie cerebral autoregulation during sleep. Lambs, instrumented to measure CBF (flow probe on the superior sagittal sinus), sleep state, and cerebral perfusion pressure (CPP), were rapidly made hypotensive by inflating a cuff around the brachiocephalic artery to reduce CPP to 30 mm Hg in each state. During control periods, cerebral vascular resistance (CVR in mm Hg/mL/min) was lower in active sleep (2.8 +/- 0.3, mean +/- SD, P < or = 0.001) than in wakefulness (3.9 +/- 0.6) and quiet sleep (4.3 +/- 0.6). The CVR decreased promptly in each state as CPP was lowered. The time (seconds) required for maximal cerebral vasodilation to occur was longer in active sleep (35 +/- 11) than in quiet sleep (20 +/- 6, P < or = 0.001) and wakefulness (27 +/- 11, P < or = 0.05). The CVR decreased less in active sleep (0.6 +/- 0.3, P < or = 0.001) than in quiet sleep (1.5 +/- 0.3), although the changes in CPP induced with brachiocephalic occlusion were equal in each state. In conclusion, our studies provide the first evidence that the vasoactive mechanisms that underlie autoregulation of the cerebral circulation function during sleep. Moreover, our data reveal that the speed and the magnitude of the vasodilatory reserves available for autoregulation are significantly less in active sleep than in quiet sleep.