Newborn Cerebral Circulation Research Articles

Hydrogen sulfide and cerebral microvascular tone in newborn pigs.

Hydrogen sulfide (H2S) is a gaseous signaling molecule that appears to be involved in numerous biological processes, including regulation of blood pressure and vascular tone. The present study is designed to address the hypothesis that H2S is a functionally significant, endogenous dilator in the newborn cerebrovascular circulation. In vivo experiments were conducted using newborn pigs with surgically implanted, closed, cranial windows. Topical application of H2S concentration-dependently (10(-6) to 2×10(-4) M) dilated pial arterioles. This dilation was blocked by glibenclamide (10(-6) M). L-cysteine, the substrate of the H2S-producing enzymes cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), also dilated pial arterioles. The dilation to L-cysteine was blocked by the CSE inhibitor d,l-propargylglycine (PPG, 10 mM) but was unaffected by the CBS inhibitor amino-oxyacetate (AOA, 1 mM). Western blots detected CSE, but not CBS, in cerebral microvessels, whereas CBS is detected in brain parenchyma. Immunohistological CSE expression is predominantly vascular while CBS is expressed mainly in neurons and astrocytes. L-cysteine (5 mM) increased H2S concentration in cerebrospinal fluid (CSF), measured by GC-MS, from 561±205 to 2,783±818 nM before but not during treatment with PPG (1,030±70 to 622±78 nM). Dilation to hypercapnia was inhibited by PPG but not AOA. Hypercapnia increased CSF H2S concentration from 763±243 to 4,337±1789 nM before but not during PPG treatment (357±178 vs. 425±217 nM). These data show that H2S is a dilator of the newborn cerebral circulation and that endogenous CSE can produce sufficient H2S to decrease vascular tone. H2S appears to be a physiologically significant dilator in the cerebral circulation.

Role of cAMP and cGMP in arachidonic acid and PGE2 ‐induced cerebral vasodilation in newborn pigs

Arachidonic acid (AA) metabolites of cyclooxygenase (COX) are important regulators of newborn cerebral circulation. PGE2 stimulates cAMP in cultured vascular smooth muscle cells. PGE2 increases cAMP and cGMP. Therefore, AA and PGE2, generated in cerebral vessels may produce dilation by increasing cGMP and cAMP. In this study we address the hypothesis that cAMP and/or cGMP mediate dilation produced by AA and PGE2. Experiments used anesthetized newborn pigs with closed cranial windows. In piglets with PGE2, indomethacin (5mg/k g iv) was administered to inhibit COX. Pial arteriolar responses to AA (10−7 M, 10−5 M), PGE2 (10−7 M, 10−5 M) or vehicle with guanylyl cyclase inhibitor ODQ (2.5 x10−5 M) or adenylyl cyclase inhibitor SQ22536 (10−6 M), or their vehicles were determined. ODQ (2.5 x10−5 M) blocked AA‐ and PGE2‐induced increases in cerebral vasodilation in newborn pigs. SQ 22536 blocked dilation produced by the adenylyl cyclase activator forskolin (10−7M) and also inhibited AA‐ and PGE2‐induced cerebral vasodilation. Both cGMP and cAMP appear to contribute to the dilator effect of AA and PGE2. Since both ODQ and SQ22536 blocked dilations to AA and PGE2, cAMP and cGMP may act in a sequential manner. Research supported by NIH/NHLBI.

Role of cAMP and cGMP in arachidonic acid and PGE2 ‐induced cerebral vasodilation in piglets

Arachidonic acid (AA) products are important contributors to regulation of newborn cerebral circulation. PGE2 stimulates cAMP production in cultured piglet cerebrovascular smooth muscle cells. PGE2 increases cAMP and cGMP in CSF. Therefore, it is possible that AA and its metabolite, PGE2, generated in cerebral vessels produce vasodilation by increasing cGMP and cAMP production. The aim of this study was to determine if cAMP and/or cGMP mediate the vasodilatory effects of AA and PGE2. Experiments were performed in anesthetized piglets with closed cranial windows. In experiments with PGE2, piglets were administered indomethacin (5mg/kg, iv) to inhibit endogenous prostanoid production. Pial arteriolar responses to topical application of AA (10−7 M, 10−5 M), PGE2 (10−7 M, 10−5 M) or vehicle were determined without and with the guanylyl cyclase inhibitor ODQ (10−5 M) or the adenylyl cyclase inhibitor SQ22536 (10−6 M). ODQ (2.5x10−5 M) blocked AA‐ and PGE2‐induced cerebral vasodilation. SQ22536, that blocked the dilation to the adenylyl cyclase activator, Forskolin (10−7 M), also inhibited AA‐ and PGE2‐induced cerebral vasodilation. These data suggest that both cGMP and cAMP mediate vasodilator effects of AA and PGE2. Since both ODQ and SQ22536 blocked the cerebral vasodilator effect of AA and PGE2, cAMP and cGMP may act in a sequential manner. Studies to address this subject are in progress.

Carbon monoxide as an attenuator of vasoconstriction in piglet cerebral arterioles.

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral circulation. The present study addressed the hypothesis that endogenous CO attenuates pial arteriolar vasoconstriction caused by hypocapnia, platelet activating factor, and elevated blood pressure. Experiments used anesthetized piglets with implanted, closed cranial windows. Topical application of a metal porphyrin inhibitor of heme oxygenase was used to inhibit production of CO. Chromium mesopophyrin increased vasoconstriction in response to hypocapnia. The constrictor response to a topical stimulus, platelet activating factor, was also increased by application of chromium mesoporphyrin. Inhibition of heme oxygenase did not constrict pial arterioles in normotensive newborn pigs (mean arterial pressure of about 70 mmHg), but did constrict pial arterioles of piglets with experimentally induced increases in arterial pressure (mean arterial pressure greater than 90 mmHg). In fact, pial arterioles of normotensive piglets transiently dilated to chromium mesoporphyrin, whereas those of hypertensive piglets progressively constricted during 10 min of chromium mesoporphyrin treatment. Therefore, inhibition of heme oxygenase augments cerebral vasoconstriction in response to several very different constrictor stimuli. These data suggest endogenous CO attenuates vasoconstrictor responses in the newborn cerebral circulation.

EFFECT OF INHIBITORS OF PROTEIN KINASE C(PKC) AND PROTEIN TYROSINE KINASE(PTK) ON THE CEREBROVASCULAR RESPONSE TO ADENOSINE 2276

Adenosine is an important factor in cerebrovascular regulation in the newborn especially under conditions low cerebral oxygen availability. Post receptor signaling associated with adenosine receptor(s) in the newborn cerebral circulation are poorly understood. Recent studies suggest that certain adenosine receptors are coupled to PKC-and/or PTK-dependent signal transduction pathways. We evaluated the hypothesis that activation of PKC modulates the cerebrovascular response to adenosine in vivo by examining the effect of staurosporine, an inhibitor of PKC, using the closed cranial window preparation in twelve newborn piglets. In that paradigm, staurosporine (9 nM) treatment augmented by 4-fold the increase pial arteriolar diameter induced by 1 μM exogenous adenosine (Δdiameter control = 4±2%[79±4 μm to 82±3 μm]; Δdiameter staurosporine = 17±3%[56±3 μm to 101±5 μm]). In addition, the adenosine-induced vasodilation was prolonged in staurosporine-treated preparations. Recent experiments show a similar effect, i.e., enhanced the dose-dependent relaxation response to adenosine in isolated, newborn lamb middle cerebral artery (MCA) preparations in tissue bath. An inhibitor of PTK (genistein, 20 μM) showed similar trend but was not statistically significant. The data suggest that activation of staurosporine-sensitive signal transduction pathways such as PKC may play a role in the ultimate expression of contractile properties of cerebral arteries from newborn animal models. We propose that certain adenosine receptors activate PKC which modulate the contraction/relaxation response and/or modify adenosine uptake and metabolism. (Supported by the American Heart Association national council, #95009270)

VASCULAR SMOOTH MUSCLE SIGNALS UPREGULATE ENDOTHELIAL CYCLOOXYGENASE EXPRESSION IN THE NEWBORN PIG CEREBRAL CIRCULATION. † 1403

Cellular communication between vascular endothelium and smooth muscle are important in the regulation of vascular function. In the newborn cerebral circulation, endothelium-derived cyclooxygenase products participate in vascular smooth muscle responsiveness to many physiological stimuli. Our present data indicate that vascular smooth muscle, in turn, may influence functions of vascular endothelium. Endothelial cells from the newborn pig cerebral microvessels cultured separately or in coculture with microvascular smooth muscle cells were used to investigate a possible role of cellular communication in the regulation of endothelial cyclooxygenase expression. Microvascular endothelial cells (freshly isolated as well as confluent and quiescent primary cultures) constitutively express both cyclooxygenase isoforms, COX-1 and COX-2, as detected by Western blots and immunostaining with the isozyme-specific antibodies. Results with the isozyme-selective inhibitor NS-398 suggest that COX-2 provides a large portion of endothelial prostanoid production. Endothelial cells grown in non-contact coculture with smooth muscle cells for 24-48 hr show induction of COX-1 and COX-2 proteins. There are a 10-50-fold increases in endogenous prostanoid accumulation and cyclooxygenase activity that are inhibited by dexamethasone, cycloheximide and actinomycin D. Similar changes are induced in endothelial cells exposed to smooth muscle cell-conditioned media. These results indicate that cerebral microvascular smooth muscle cells produce a diffusable compound(s) that upregulate endothelial cyclooxygenase thus modifying endothelial cell function.

Functional study on vasodilator effects of prostaglandin E 2 in the newborn pig cerebral circulation

Cerebral vascular reactivity to prostaglandin E 2 was investigated in newborn pigs using closed cranial windows. Exogenous prostaglandin E 2 is a dilator of pial arterioles that elevates cyclic AMP in cortical cerebrospinal fluid. Pial arterioles are less sensitive to prostaglandin E 2 than to the prostacyclin receptor agonist iloprost, but their maximal responses to the dilator prostanoids are similar. The cerebrovascular effects of prostaglandin E 2 and iloprost are not additive. Pretreatment with either iloprost or prostaglandin E 2 decreases pial arteriolar responsiveness to iloprost without affecting responses to isoproterenol. The homologous desensitization of pial arterioles suggests that auto- and cross-tachyphylaxis in vascular effects of iloprost and prostaglandin E 2 occur at the receptor level. Indomethacin, which selectively inhibits prostacyclin receptor-mediated responses in cerebral vascular smooth muscle, greatly reduces the vascular responses to prostaglandin E 2. These results suggest that vasodilator effects of prostaglandin E 2 in the newborn cerebral circulation are mediated via prostacyclin receptors coupled to adenylyl cyclase.

Response of the Newborn Cerebral Circulation to Cocaine

Cocaine abuse by pregnant women is often associated with neurological injury in the newborn. To explore a vascular-related mechanism of injury, we investigated the effect of cocaine on the cerebral circulation in newborn pigs. During normoxic conditions, cocaine administration (1.5 mg/kg i.v.), resulting in peak plasma cocaine levels on the order of 10(-6) M, decreased cerebral blood flow (CBF) by 14%, as measured by the tracer microsphere method. To elicit the mechanisms by which cocaine decreased CBF, closed cranial windows were placed and the diameter of pial arterioles was measured by intravital microscopy while cocaine (10(-6) M) was applied onto the cortical surface. Topically applied cocaine decreased pial arteriolar diameter by 9%. Vasoconstriction induced by topically applied cocaine was blocked by tetrodotoxin (10(-7) M, Na+ channel blocker), whereas phentolamine (10(-5) M, noradrenergic receptor blocker) had no effect on the arteriolar response to cocaine, which suggested that cocaine effected constriction by an anesthetic and not a sympathomimetic mechanism. To evaluate this hypothesis further, cerebral vessels in the right hemibrain were sympathetically denervated while those in the left hemibrain remained innervated. During normoxia, cocaine (1.5 mg/kg i.v.) decreased CBF equally in both hemibrains, confirming the non-sympathomimetic mechanism. During asphyxia, cocaine administration attenuated cerebral hyperemia in both hemibrains, but in innervated more than in denervated, indicating that anesthetic and sympathomimetic vasoconstriction occurred during asphyxia. We conclude that cocaine constricts the immature cerebrovasculature and decreases CBF by an anesthetic mechanism during normoxic conditions and by both sympathomimetic and anesthetic mechanisms during asphyxia.

Effects of indomethacin on cerebral vasodilator responses to arachidonic acid and hypercapnia in newborn pigs.

Responses of pial arterioles to topically applied arachidonic acid, conversion of exogenous arachidonic acid to prostanoids, and pial arteriolar dilation to hypercapnia were examined before and at progressive times after treatment with indomethacin (5 mg/kg i.v.) in chloralose-anesthetized newborn pigs with closed cranial windows. Before treatment with indomethacin, arachidonic acid and hypercapnia dilated pial arterioles and increased cortical periarachnoid cerebrospinal fluid concentrations of 6-keto-prostaglandin (PG) F1 alpha and PGE2. One h after indomethacin treatment, the dilations and prostanoid synthesis were blocked. By 2 h after indomethacin treatment, hypercapnia produced significant dilation of pial arterioles, and dilation to both stimuli had returned to preindomethacin levels by 3 h. Inhibition of conversion of exogenous arachidonic acid to prostanoids as monitored by increases in 6-keto-PGF1 alpha and PGE2 in cerebrospinal fluid under the window also was reversed by 3 h after treatment with indomethacin. Repeated indomethacin treatment again blocked dilations and conversion of arachidonic acid to prostanoids on the brain surface. The possibility of short duration of vascular effectiveness of indomethacin when it is administered systemically needs to be considered, both when it is used as a probe for understanding contributions of PGH synthase products to control of cerebral circulation and when it is used therapeutically in attempts to alter the newborn cerebral circulation.