Biomedical subjects

D A Pelligrino

Publications and source records attributed to D A Pelligrino.

At least 19 recordsLinked to original sources

PubMed2004

Influence of the glia limitans on pial arteriolar relaxation in the rat.

We examined whether damage to the glia limitans (GL), via exposure to the gliotoxin l-alpha-aminoadipic acid (l-alphaAAA), alters hypercapnia-induced pial arteriolar dilation in vivo. Anesthetized female rats were prepared with closed cranial windows. Pial arteriolar diameters were measured using intravital microscopy. l-alphaAAA (2 mM) was injected into the space under the cranial windows 24 h before the study, and injury to the GL was confirmed by light microscopy. l-alphaAAA was associated with a reduction in pial arteriolar CO(2) reactivity to 40-50% of the level seen in vehicle-treated controls, with no further reduction in the CO(2) response after nitric oxide (NO) synthase (NOS) inhibition via N(omega)-nitro-l-arginine (l-NNA). Subsequent blockade of prostanoid synthesis, via indomethacin (Indo), reduced CO(2) reactivity to 10-15% of normal. In vehicle-treated controls, l-NNA, followed by Indo, reduced the response to approximately 50% and then to 15-20% of the normocapnic value, respectively. On the other hand, l-alphaAAA had no effect on vascular responses to the endothelium-dependent vasodilator acetylcholine or the NO donor SNAP and did not alter cortical somatosensory evoked responses. This indicates an absence of any direct l-alphaAAA actions on pial arterioles or influence on neuronal transmission. Furthermore, l-alphaAAA did not alter the vasodilation elicited by topical application of an acidic artificial cerebrospinal fluid solution, suggesting that the GL influences the pial arteriolar relaxation elicited by hypercapnic, but not local extracellular (EC), acidosis. That differences exist in the mechanisms mediating hypercapnia- versus EC acidosis-induced pial arteriolar dilations was further exemplified by the finding that topical application of a neuronal NOS (nNOS)-selective blocker (ARR-17477) reduced the response to hypercapnia (by approximately 65%) but not the response to EC acidosis. Disruption of GL gap junctional communication, using an antisense oligodeoxynucleotide (ODN) connexin43 knockdown approach, was accompanied by a 33% lower CO(2) reactivity versus missense ODN-treated controls. These results suggest that the GL contribution to the hypercapnic vascular response appears to involve the NO-dependent component rather than the prostanoid-dependent component and may involve gap junctional communication. We speculate that the GL may act to facilitate the spread, to pial vessels, of hypercapnia-induced vasodilating signals arising in the comparatively few scattered nNOS neurons that lie well beneath the GL.

2-Aminoadipic Acid↗

PubMed2003

Nascent EDHF-mediated cerebral vasodilation in ovariectomized rats is not induced by eNOS dysfunction.

In estrogen-depleted [i.e., ovariectomized (Ovx)] animals, an endothelium-derived hyperpolarizing factor (EDHF)-like mechanism may arise to, at least partially, replace endothelial nitric oxide (NO) synthase (eNOS)-derived NO in modulating cerebral arteriolar tone. Additional findings show that eNOS expression and function is restored in estrogen-treated Ovx female rats, while the nascent EDHF-like activity disappears. Because NO has been linked to repression of EDHF activity in the periphery, the current study was undertaken to examine whether the nascent EDHF role in cerebral vessels of Ovx females relates to a chronically repressed eNOS-derived NO-generating function. We compared the effects of chronic NOS inhibition with Nomega-nitro-L-arginine-methyl ester (L-NAME; 100 mg. kg-1. day-1 for 3 wk) on EDHF-mediated pial arteriolar vasodilation in anesthetized intact, Ovx, and 17beta-estradiol-treated (0.1 mg. kg-1. day-1 ip, 1 wk) Ovx (OVE) female rats as well as in male rats that were prepared with closed cranial windows. In the chronic NOS inhibition groups, pial arteriolar responses were monitored in the absence (all groups) and presence (females only) of indomethacin (Indo; 10 mg/kg iv). Finally, the gap junction inhibitory peptide Gap 27 (300 muM) was applied to block EDHF-related vasodilation. NO donor (S-nitroso-N-acetyl-penicillamine) responses were similar in all rats studied. Acetylcholine (ACh) reactivity was virtually absent in control Ovx rats and chronically NOS-inhibited intact female, OVE, and male rats. However, a partial recovery of ACh reactivity was seen in L-NAME-treated Ovx females. In addition, in the presence of L-NAME, a normal CO2 reactivity was observed in all females, whereas a 50% reduction in CO2 reactivity was seen in males. In intact and OVE rats, both chronic and acute (NG-nitro-L-arginine suffusion) NOS inhibition, combined with Indo, depressed ADP-induced dilation by > or =50%, and subsequent application of Gap 27 had no further effect on ADP-induced vasodilation. ADP reactivity was retained in Ovx rats after combined chronic NOS inhibition and acute Indo, but was attenuated significantly by Gap 27. In males, Gap 27 had no effect on arteriolar reactivity. Taken together, our data demonstrate that in the cerebral microcirculation, NO does not have an inhibitory effect on EDHF production or action. The increased EDHF-like function in chronic estrogen-depleted animals is not due to eNOS deficiency, suggesting a more direct effect of estrogen in modulating EDHF-mediated cerebral vasodilation.

Acetylcholine↗

PubMed2002

ADP-induced pial arteriolar dilation in ovariectomized rats involves gap junctional communication.

It was previously shown that, despite the loss of nitric oxide (NO) dependence, ADP-induced pial arteriolar dilation was not attenuated in estrogen-depleted [i.e., ovariectomized (Ovx)] rats. Additional evidence suggested that the NO was replaced by an endothelium-dependent hyperpolarizing factor (EDHF)-like mechanism. To further characterize the nascent EDHF role in Ovx females, the current study was undertaken to test whether, in Ovx rats, ADP-induced pial arteriolar dilation retained its endothelial dependence and whether gap junctions are involved in that response. A closed cranial window and intravital microscopy system was used to monitor pial arteriolar diameter changes in anesthetized rats. The endothelial portion of the ADP-induced dilation was evaluated using light dye endothelial injury (L/D). The study was organized around three experimental approaches. First, the responses of pial arterioles to ADP before and after L/D exposure in intact and Ovx female rats were tested. L/D reduced the ADP response by 50-70% in both groups, thereby indicating that the endothelium dependence of ADP-induced vasodilation is not altered by chronic estrogen depletion. Second, the NO synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) and the prostanoid synthesis inhibitor indomethacin (Indo) were coapplied. In intact females, L-NNA-Indo attenuated the response to ADP by 50%, with no further changes upon the addition of L/D. On the other hand, L-NNA-Indo did not affect ADP reactivity in Ovx rats, but subsequent L/D exposure reduced the ADP response by >50%. The NO-prostanoid-independent, but endothelium-dependent, nature of the response in Ovx females is a hallmark of EDHF participation. Third, gap junctional inhibition strategies were applied. A selective inhibitor of gap junctional function, Gap 27, did not affect ADP reactivity in intact females but reduced the the ADP response by 50% in Ovx females. A similar result was obtained following application of a connexin43 antisense oligonucleotide. These findings suggest that the nascent EDHF dependency of ADP-induced pial arteriolar dilation in Ovx females involves connexin43-related gap junctional communication.

Adenosine Diphosphate↗

PubMed2002

Agonist-specific differences in mechanisms mediating eNOS-dependent pial arteriolar dilation in rats.

Nitric oxide (NO), derived from the endothelial isoform of NO synthase (eNOS), is a vital mediator of cerebral vasodilation. In the present study, we addressed the issue of whether the mechanisms responsible for agonist-induced eNOS activation differ according to the specific receptor being stimulated. Thus we examined whether heat shock protein 90 (HSP90), phosphatidylinositol-3-kinase (PI3K), and tyrosine kinase participate in ACh- versus ADP-induced eNOS activation in cerebral arterioles in vivo. Pial arteriolar diameter changes in anesthetized male rats were measured during sequential applications of ACh and ADP in the absence and presence of the nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME), the neuronal NOS (nNOS)-selective inhibitor ARR-17477, the HSP90 blocker 17-(allylamino)-17-demethoxygeldanamycin (AAG), the PI3K inhibitor wortmannin (Wort), or the tyrosine kinase blocker tyrphostin 47 (T-47). Only NOS inhibition with L-NAME (not ARR-17477) reduced ACh and ADP responses (by 65-75%), which suggests that all of the NO dependence in the vasodilating actions of those agonists derived from eNOS. Suffusions of AAG, Wort, and T-47 were accompanied by substantial reductions in ACh-induced dilations but no changes in the responses to ADP. These findings suggest that muscarinic (ACh) and purinergic (ADP) receptor-mediated eNOS activation in cerebral arterioles involve distinctly different signal transduction pathways.

Acetylcholine↗

PubMed2001

The key role of caveolin-1 in estrogen-mediated regulation of endothelial nitric oxide synthase function in cerebral arterioles in vivo.

The marked impairment in cerebrovascular endothelial nitric oxide synthase (eNOS) function that develops after ovariectomy may relate to the observation that the abundance of cerebral vascular eNOS and its endogenous inhibitor, caveolin-1, vary in opposite directions with chronic changes in estrogen status. The authors endeavored, therefore, to establish a link between these correlative findings by independently manipulating, in ovariectomized female rats, eNOS and caveolin-1 expression, while monitoring agonist (acetylcholine)-stimulated eNOS functional activity. In the current study, the authors showed that individually neither the up-regulation of eNOS (through simvastatin treatment), nor the down-regulation of caveolin-1 (through antisense oligonucleotide administration) is capable of restoring eNOS function in pial arterioles in vivo in these estrogen-depleted rats. Only when eNOS up-regulation and caveolin-1 down-regulation are combined is activity normalized. These results establish a mechanistic link between the estrogen-associated divergent changes in the abundance of caveolin-1 and eNOS protein and eNOS functional activity in cerebral arterioles.

Acetylcholine↗

PubMed2001

beta-Adrenoceptor and nNOS-derived NO interactions modulate hypoglycemic pial arteriolar dilation in rats.

We examined the relative contributions from nitric oxide (NO) and catecholaminergic pathways in promoting cerebral arteriolar dilation during hypoglycemia (plasma glucose congruent with 1.4 mM). To that end, we monitored the effects of beta-adrenoceptor (beta-AR) blockade with propranolol (Pro, 1.5 mg/kg iv), neuronal nitric oxide synthase (nNOS) inhibition with 7-nitroindazole (7-NI, 40 mg/kg ip) or ARR-17477 (300 microM, via topical application), or combined intravenous Pro + 7-NI or ARR-17477 on pial arteriolar diameter changes in anesthetized rats subjected to insulin-induced hypoglycemia. Additional experiments, employing topically applied TTX (1 microM), addressed the possibility that the pial arteriolar response to hypoglycemia required neuronal transmission. Separately, Pro and 7-NI elicited modest but statistically insignificant 10-20% reductions in the normal ~40% increase in arteriolar diameter accompanying hypoglycemia. However, combined Pro-7-NI was accompanied by a >80% reduction in the hypoglycemia-induced dilation. On the other hand, the combination of intravenous Pro and topical ARR-17477 did not affect the hypoglycemia response. In the presence of TTX, the pial arteriolar response to hypoglycemia was lost completely. These results suggest that 1) beta-ARs and nNOS-derived NO interact in contributing to hypoglycemia-induced pial arteriolar dilation; 2) the interaction does not occur in the vicinity of the arteriole; and 3) the vasodilating signal is transmitted via a neuronal pathway.

Action Potentials↗

PubMed2001

Chronic estrogen depletion alters adenosine diphosphate-induced pial arteriolar dilation in female rats.

We examined pial arteriolar reactivity to a partially endothelial nitric oxide synthase (eNOS)-dependent vasodilator ADP as a function of chronic estrogen status. The eNOS-dependent portion of the ADP response was ascertained by comparing ADP-induced pial arteriolar dilations before and after suffusion of a NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA; 1 mM) in intact, ovariectomized (Ovx), and 17beta-estradiol (E2)-treated Ovx females. We also examined whether ovariectomy altered the participation of other factors in the ADP response. Those factors were the following: 1) the prostanoid indomethacin (Indo); 2) the Ca2+-dependent K+ (K(Ca)) channel, iberiotoxin (IbTX); 3) the ATP-regulated K+ (K(ATP)) channel glibenclamide (Glib); 4) the K(Ca)-regulating epoxygenase pathway miconazole (Mic); and 5) the adenosine receptor 8-sulfophenyltheophylline (8-SPT). In intact females, the eNOS-dependent (L-NNA sensitive) portion of the ADP response represented approximately 50% of the total. The ADP response was retained in the Ovx rats but L-NNA sensitivity disappeared. On E2 replacement, the initial pattern was restored. ADP reactivity was unaffected by Indo, Glib, Mic, and 8-SPT. IbTX was associated with 50-80% reductions in the response to ADP in the intact group that was nonadditive with L-NNA, and 60-100% reductions in the Ovx group. The present findings suggest that estrogen influences the mechanisms responsible for ADP-induced vasodilation. The continued sensitivity to IbTX in Ovx rats, despite the loss of a NO contribution, is suggestive of a conversion to a hyperpolarizing factor dependency in the absence of E2.

Adenosine Diphosphate↗

PubMed2001

Estrogen and cerebrovascular physiology and pathophysiology.

Numerous studies have uncovered a wide variety of estrogen effects with apparent cardiovascular benefits, the most recognized ones being vasodilation, anti-atherogenesis, diminished post-ischemic inflammation and anti-oxidant effects. This article provides an overview of the influence of estrogen on the cerebral vasculature, under physiologic and pathophysiologic conditions, and covers both acute and chronic effects. The discussion is primarily focused on the vasodilatory and anti-inflammatory actions of estrogen, since those particular estrogen influences have received the greatest attention in studies published to date. With respect to vasodilation, although some consideration is given to the role of other vasodilating mechanisms and factors, the emphasis is mostly placed on the endothelial isoform of nitric oxide synthase, eNOS, which has emerged as a clear target of estrogen. Some consideration is given to recent findings that suggest that estrogen can stimulate eNOS activity by decreasing the expression of the eNOS inhibitor caveolin-1. With regard to the ability of estrogen to counteract inflammation, potential mechanisms by which estrogen limits the post-ischemic leukocyte adhesion, and the expression of the inducible NOS, are discussed.

Animals↗

PubMed2000

Relative contributions from neuronal and endothelial nitric oxide synthases to regional cerebral blood flow changes during forebrain ischemia in rats.

The principal aim of this study was to examine the relative contributions from the neuronal and endothelial isoforms of nitric oxide synthase (nNOS and eNOS, respectively) in their capacity to modulate intra-ischemic cerebral blood flow (CBF) changes, in the ischemically vulnerable hippocampus and striatum. CBF changes were monitored, using laser-Doppler flowmetry, in rats subjected to 30 min of forebrain ischemia (right common carotid occlusion+hemorrhagic hypotension). Rats were pretreated with a selective nNOS inhibitor (ARR 17477), a NOS inhibitor that blocks both eNOS and nNOS (N(G)-nitro-L-arginine; L-NNA), or saline (control). In initial experiments, where ischemic MABP was targeted to exactly 30 mmHg, NOS inhibition reduced intra-ischemic cortical CBF from the control level of approximately 20% of baseline to 3% (L-NNA) or 6% (ARR 17477) of baseline. The statistically similar effects of the two NOS inhibitors confirmed that nNOS is the predominant NO source supporting intra-ischemic vasodilation in the cortex. In subsequent experiments, CBF was measured in the right hippocampus, and striatum, as well as the cortex, and, to reduce data variability, blood withdrawal was adjusted to achieve an intra-ischemic cortical CBF of 20% (controls) or 5% (NOS inhibited rats) of baseline. In those groups, mean ischemic MABP levels ranged from 28 to 32 mmHg. In controls, intra-ischemic CBF fell to 20%, 45%, and 47% of baseline in the cortex, hippocampus, and striatum, respectively. With nNOS inhibition, intra-ischemic CBF was further reduced to 5%, 15%, and 18% of baseline, respectively. However, with combined eNOS/nNOS inhibition, the CBF values were 5%, 37%, and 21%, respectively. These results suggest that the nNOS contribution to intra-ischemic vasodilation in vulnerable regions is substantially greater than eNOS. The significantly higher intra-ischemic CBF level in the hippocampus in combined eNOS/nNOS vs nNOS-inhibited rats may relate, in contrast to other regions, to a low eNOS influence on vascular function in that structure and CBF redistribution to the hippocampus when eNOS activity is blocked globally.

Amidines↗

PubMed2000

Nitric-oxide-dependent pial arteriolar dilation in the female rat: effects of chronic estrogen depletion and repletion.

In this study, we compared endothelial nitric oxide synthase (eNOS)-mediated cerebral vasodilating responses in intact female rats, chronically ovariectomized (OVX) rats, and OVX rats treated for 2 weeks with 17beta-estradiol (E(2)). Under anesthesia, using intravital microscopy and a closed cranial window system, pial arteriolar diameter changes were monitored during sequential cortical suffusions of an eNOS-dependent dilator [acetylcholine (ACh)] and a direct NO donor [S-nitrosoacetylpenicillamine (SNAP)]. In separate rats from the same groups, we compared eNOS and caveolin-1 (CAV-1) protein abundance in pial arterioles (via immunofluorescence analyses). In untreated and low-dose E(2)-treated (1.0 microg x kg(-1) x day(-1)) OVX rats, ACh-induced vasodilations were virtually absent. High-dose E(2) treatment (100 microg x kg(-1) x day(-1)) restored ACh-induced pial arteriolar dilations to levels seen in intact females. The vasodilations elicited by SNAP and ADO were unaffected by chronic estrogen changes, indicating no direct estrogen influence on vascular smooth muscle (VSM) reactivity. Pial arteriolar eNOS protein abundance was diminished by ovariectomy and restored by high-dose E(2) treatment. Pial arteriolar CAV-1 expression was higher in OVX versus intact and E(2)-treated OVX females. These results suggest that long-term changes in estrogen directly influence brain eNOS functional activity. The estrogen-related changes in eNOS-dependent vasodilating function appear to be related, in part, to a capacity for E(2) to increase eNOS protein expression and, in part, to an E(2)-associated diminution in endothelial CAV-1 expression.

Acetylcholine↗

PubMed2000

Cyclic adenosine monophosphate regulates the expression of the intercellular adhesion molecule and the inducible nitric oxide synthase in brain endothelial cells.

The authors studied whether cyclic AMP (cAMP), a widespread regulator of inflammation, modulates the cytokine-mediated expression of the intercellular adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and the inflammatory nitric oxide synthase 2 (NOS-2), in primary and immortalized brain endothelial cell cultures (GP8.3 cell line). When measured by enzyme-linked immunosorbent assay (ELISA), ICAM-1 was constitutively expressed and was up-regulated twofold by interleukin-1beta, with no effect of interferon-gamma. The NOS-2 activity, assessed by nitrite accumulation, was absent from untreated cultures but was induced by interleukin-1beta and interferon-gamma acting synergistically. Stimulation of cAMP-dependent pathways with forskolin or dibutyryl cAMP decreased ICAM-1 protein expression, whereas it increased NOS-2 protein expression. For both ICAM-1 and NOS-2, mRNA expression correlated with protein expression. Blockade of NOS activity with L-N-monomethylargiuine (L-NMMA) did not alter ICAM-1 expression, indicating that the nitric oxide released by NOS-2 did not cause the down-regulation of ICAM-1. Analysis of NFKB activation indicated that cAMP acted through a mechanism other than inhibition of nuclear translocation of NFKB. The authors conclude that cAMP modulates the expression of proinflammatory molecules in brain endothelium. This suggests that inflammatory processes at the blood-brain barrier in vivo may be regulated by perivascular neurotransmitters via cAMP.

Animals↗

PubMed2000

Effects of estrogen on leukocyte adhesion after transient forebrain ischemia.

BACKGROUND AND PURPOSE: Recent findings indicate that estrogen (ie, 17beta-estradiol [E(2)]) provides neuroprotection in models of transient global and focal ischemia. Enhanced postischemic leukocyte adhesion and infiltration have been linked to neuropathology in the brain as well as other tissues. We recently showed that estrogen reduces leukocyte adhesion in the cerebral circulation of female rats during resting conditions. METHODS: We compared leukocyte adhesion in pial venules in vivo in intact, ovariectomized (OVX), and E(2)-treated OVX female rats subjected to transient forebrain ischemia (30-minute right common carotid artery occlusion and hemorrhagic hypotension) and reperfusion. Adherent rhodamine-6G-labeled leukocytes were viewed through a closed cranial window with the use of intravital microscopy. Leukocyte adhesion was measured before ischemia and at different times after reperfusion. RESULTS: Before ischemia, leukocyte adhesion (measured as a percentage of venular area occupied by adherent leukocytes) was 2 to 3 times greater in OVX versus intact or E(2)-treated OVX rats (7.0%, 3.4%, and 2.2%, respectively). This difference disappeared at 120 minutes of reperfusion, when comparable levels of enhanced leukocyte adhesion were observed in all groups. In OVX rats, leukocyte adhesion remained elevated after 4 and 6 hours of reperfusion (11.6% and 12.9%, respectively), while the other 2 groups showed significantly lower levels (5.0% and 5.8% for intact rats and 7.0% and 7.2% for E(2)-treated OVX rats). CONCLUSIONS: Present results demonstrate that estrogen modulates leukocyte adhesion in the cerebral circulation after transient forebrain ischemia. This effect suggests that decreased leukocyte adhesion may be an important mechanism in estrogen-mediated neuroprotection.

Animals↗

PubMed1999

Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning.

Neuroprotection against cerebral ischemia can be realized if the brain is preconditioned by previous exposure to a brief period of sublethal ischemia. The present study was undertaken to test the hypothesis that nitric oxide (NO) produced from the neuronal isoform of NO synthase (NOS) serves as a necessary signal for establishing an ischemia-tolerant state in brain. A newborn rat model of hypoxic preconditioning was used, wherein exposure to sublethal hypoxia (8% oxygen) for 3 hours renders postnatal day (PND) 6 animals completely resistant to a cerebral hypoxic-ischemic insult imposed 24 hours later. Postnatal day 6 animals were treated 0.5 hour before preconditioning hypoxia with the nonselective NOS inhibitor L-nitroarginine (2 mg/kg intraperitoneally). This treatment, which resulted in a 67 to 81% inhibition of calcium-dependent constitutive NOS activity 0.5 to 3.5 hours after its administration, completely blocked preconditioning-induced protection. However, administration of the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg intraperitoneally) before preconditioning hypoxia, which decreased constitutive brain NOS activity by 58 to 81%, was without effect on preconditioning-induced cerebroprotection, as was pretreatment with the inducible NOS inhibitor aminoguanidine (400 mg/kg intraperitoneally). The protective effects of preconditioning were also not blocked by treating animals with competitive [3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate; 5 mg/kg intraperitoneally] or noncompetitive (MK-801; 1 mg/kg intraperitoneally) N-methyl-D-aspartate receptor antagonists prior to preconditioning hypoxia. These findings indicate that NO production and activity are critical to the induction of ischemic tolerance in this model. However, the results argue against the involvement of the neuronal NOS isoform, activated secondary to a hypoxia-induced stimulation of N-methyl-D-aspartate receptors, and against the involvement of the inducible NOS isoform, but rather suggest that NO produced by the endothelial NOS isoform is required to mediate this profound protective effect.

Animals↗

PubMed1999

Estrogen reduces leukocyte adhesion in the cerebral circulation of female rats.

In this study the authors addressed the hypothesis that estrogen (i.e., 17beta-estradiol) acts to repress leukocyte adhesion. The experiments involved comparing leukocyte adhesion in cerebral venules in vivo, in intact ovariectomized and 17beta-estradiol-treated (100 microg/kg/day for 1 week) ovariectomized female rats using topical applications of the adhesion-promoting drug, phorbol 12-myristate 13-acetate (PMA). Adherent Rhodamine-6G-labeled leukocytes were viewed through a closed cranial window using intravital microscopy/videometry. Leukocyte dynamics were recorded at baseline and after each dose of PMA. The PMA was suffused (1.0 mL/min) at increasing concentrations (0.01, 0.1, and 1.0 micromol/L, 15 minutes at each level). A videotape record of each experiment was made for subsequent analysis of leukocyte adhesion. The data showed that the percentage venular area occupied by adherent leukocytes at baseline was significantly greater in the ovariectomized compared to the intact and 17beta-estradiol-treated groups (12.2%, 3.4%, and 4.2% respectively). That relationship was maintained during PMA treatments to the extent that the percentage venular area occupied by adherent leukocytes increased to 26.4% in the untreated ovariectomized group compared to 14.4% and 11.3% in the intact and 17beta-estradiol-treated groups, respectively. In conclusion, the authors found chronic estrogen depletion enhances leukocyte adhesion in the rat cerebral circulation. Estrogen repletion in such animals is accompanied by a significant reduction in leukocyte adhesion. These findings could, at least in part, account for the ischemic brain damage seen in ovariectomized versus intact females, and the restored neuroprotection observed upon 17beta-estradiol treatment reported in earlier studies.

Animals↗

PubMed1999

Possible obligatory functions of cyclic nucleotides in hypercapnia-induced cerebral vasodilation in adult rats.

Current evidence suggests that nitric oxide (NO) and vasodilating prostanoids, possibly via the actions of cGMP and cAMP, play permissive roles in hypercapnic cerebral vasodilation. The present study examined whether cGMP and cAMP have obligatory functions in hypercapnia. Using a closed cranial window in adult rats, we measured pial arteriolar diameters and periarachnoid cerebrospinal fluid (pCSF) cyclic nucleotide levels during normo- and hypercapnia and in the presence or absence of inhibitors of neuronal NO synthase (nNOS) or cyclooxygenase (COX). Also, we measured cGMP and cAMP contents in primary neuronal and astrocyte cultures, at different levels of CO2. Hypercapnia (arterial PCO2 65 mmHg)-induced pial arteriolar dilation was accompanied by 70-80% elevations in pCSF cGMP and cAMP. Inhibition of nNOS with 7-nitroindazole (7-NI) significantly reduced both the CO2-induced arteriolar dilation (by 77%) and the pCSF cGMP and cAMP increases (by 60-70%). Inhibition of COX with indomethacin reduced arteriolar CO2 reactivity (by 83%) and pCSF cyclic nucleotide increases (by 80-100%). In neuronal cultures a transient NO-dependent increase in cGMP, but not cAMP, was seen when the CO2 level was raised from 5 to 14%. No changes were seen in astrocytes. The 7-NI and indomethacin-inhibitable increases in pial arteriolar diameter and cyclic nucleotide production during hypercapnia suggest a link between these two responses. One possible, although not exclusive, interpretation of these findings is that the cyclic nucleotides have an obligatory function in the CO2 response. The large overlap in the abilities of nNOS and COX inhibitors to elicit those effects further implies interactions ("cross talk") between the cGMP and cAMP vasodilating pathways. The in vitro data suggest that hypercapnia stimulates NO production in neurons.

Animals↗

PubMed1999

Miconazole represses CO(2)-induced pial arteriolar dilation only under selected circumstances.

Previous experimental findings have led to the suggestion that guanosine 3',5'-cyclic monophosphate (cGMP) plays a permissive role in hypercapnic cerebral vasodilation. However, we recently reported that the technique used to reveal a permissive role for cGMP [cGMP repletion in the presence of nitric oxide synthase (NOS) inhibition] created a situation where CO(2) reactivity was normalized but where different mechanisms (i.e., K(+) channels) participated in the response. In the present study, we examined whether that nascent K(+)-channel dependence is related in any way to an increase in the influence of the miconazole-inhibitable cytochrome P-450 epoxygenase pathway. Using intravital microscopy and a closed cranial window system in adult rats, we measured pial arteriolar diameters during normo- and hypercapnia, first in the absence and then in the presence of a neuronal NOS (nNOS) inhibitor [7-nitroindazole (7-NI)]. This was followed by suffusion of a cGMP analog and then cGMP plus miconazole. Separate groups of rats were used to evaluate whether miconazole either alone or in the presence of 8-bromoguanosine 3', 5'-cyclic monophosphate (8-BrcGMP) or its vehicle (0.1% ethanol) had any effect on CO(2) reactivity and whether miconazole affected K(+)-channel opener-induced dilations. Hypercapnic (arterial PCO(2), congruent with65 mmHg) pial arteriolar dilations, as expected, were reduced by 70-80% with 7-NI and restored with cGMP repletion. CO(2) reactivity was again attenuated after miconazole introduction. Miconazole, with and without 8-BrcGMP, and its vehicle had no influence on pial arteriolar CO(2) reactivity in the absence of nNOS inhibition combined with cGMP repletion. Miconazole alone also did not affect vasodilatory responses to K(+)-channel openers. Thus present results suggest that the nascent K(+)-channel dependence of the hypercapnic response found in our earlier study may be related to increased epoxygenase activity. The specific reasons why the pial arteriolar CO(2) reactivity gains a K(+)-channel and epoxygenase dependence only under conditions of nNOS inhibition and cGMP restoration remain to be identified. These findings again call into question the interpretations applied to data collected in studies evaluating potential permissive actions of cGMP or NO.

Animals↗

PubMed1999

Effects of some guanidino compounds on human cerebral arteries.

BACKGROUND AND PURPOSE: Accumulation of endogenous guanidino-substituted analogues of L-arginine in chronic renal failure might contribute to some of the vascular and neurological disorders of this pathology. We tested the hypothesis that in human cerebral arteries, some guanidino compounds may increase vascular tone, through nitric oxide (NO) synthase inhibition, and impair endothelium-dependent relaxation. METHODS: Rings of human middle cerebral artery were obtained during autopsy of 26 patients who had died 3 to 12 hours before. The rings were suspended in organ baths for isometric recording of tension. We then studied the responses to N(G)-monomethyl-L-arginine (L-NMMA), N(G),N(G)-dimethyl-L-arginine (asymmetrical dimethylarginine; ADMA), aminoguanidine (AG), and methylguanidine (MG). RESULTS: L-NMMA (10(-6) to 3x10(-4) mol/L) and ADMA (10(-6) to 3x10(-4) mol/L) caused concentration- and endothelium-dependent contractions (median effective concentrations [EC(50)]=1.1x10(-5) and 1.6x10(-5) mol/L, respectively; E(max)=35. 5+/-7.9% and 43.9+/-5.9% of the response to 100 mmol/L KCl). AG (10(-5) to 3x10(-3) mol/L) and MG (10(-5) to 3x10(-3) mol/L) produced endothelium-independent contractions (E(max)=44.3+/-8.8% and 45.7+/-5.8% of the response to 100 mmol/L KCl, respectively). L-Arginine (10(-3) mol/L) prevented the contractions by L-NMMA and ADMA but did not change contractions induced by AG and MG. L-NMMA and ADMA inhibited endothelium-dependent relaxation induced by acetylcholine in a concentration-dependent manner; AG and MG were without effect. CONCLUSIONS: The results suggest that the contractions induced by L-NMMA and ADMA are due to inhibition of endothelial NO synthase activity, whereas AG and MG do not affect the synthesis of NO. An increase in the plasma concentration of L-NMMA and ADMA associated with uremia is likely to represent a diminished release or effect of NO, and consequently, an increased cerebrovascular tone in uremic patients is highly conceivable.

Adult↗

PubMed1999

Estrogen provides neuroprotection in transient forebrain ischemia through perfusion-independent mechanisms in rats.

BACKGROUND AND PURPOSE: Estrogen-related neuroprotection in association with animal models of transient forebrain and focal ischemia has been documented in several recent reports. Some of those studies indicated that part of that benefit was a function of improved intraischemic vasodilating capacity. In the present study we examined whether chronic estrogen depletion and repletion affected ischemic neuropathology through perfusion-independent mechanisms. METHODS: Normal, ovariectomized (OVX), and OVX female rats treated with 17beta-estradiol (E2) were subjected to 30 minutes of transient forebrain ischemia (right common carotid occlusion plus hemorrhagic hypotension) and reperfusion. Neurological function and brain histopathology were assessed over the 72-hour recovery period. In all rats, preischemic and intraischemic cortical cerebral blood flow (CBF) levels were monitored with laser-Doppler flowmetry. In additional rats, CBF changes in the striatum and hippocampus were also monitored with laser-Doppler flowmetry probes and radiolabeled microspheres. In each experiment, the level of ischemia was targeted to a 75% to 80% reduction in cortical CBF. RESULTS: The similarity in ischemic severity among groups was supported by measurements of comparable patterns of electroencephalographic power changes during the ischemic period. Compared with normal females, OVX rats showed diminished neurological outcomes and more severe histopathology in the hippocampus and striatum. Two-week treatment of OVX rats with E2 was accompanied by postischemic neuropathological changes similar to those seen in normal females. Intraischemic CBF reductions in the hippocampus and striatum were similar in all groups (to 35% to 50% of the preischemic value) but significantly less than the cortical CBF reductions. CONCLUSIONS: These findings indicate that estrogen provides ischemic neuroprotection through mechanisms unrelated to improvement of intraischemic cerebral perfusion.

Animals↗