Heidi M. Koenig

The role of neuronal nitric oxide synthase in regulation of cerebral blood flow in normocapnia and hypercapnia in rats

The nitric oxide synthase (NOS) inhibitors, nitro-L-arginine, its methyl ester, and N-monomethyl-L-arginine, have been shown to attenuate resting CBF and hypercapnia-induced cerebrovasodilation. Those agents nonspecifically inhibit the endothelial and neuronal NOS (eNOS and nNOS). In the present study, we used a novel nNOS inhibitor, 7-nitroindazole (7-NI) to examine the role of nNOS in CBF during normocapnia and hypercapnia in fentanyl/N2O-anesthetized rats. CBF was monitored using laser-Doppler flowmetry. Administration of 7-NI (80 mg kg−1 i.p.) reduced cortical brain NOS activity by 57%, the resting CBF by 19–27%, and the CBF response to hypercapnia by 60%. The 60% reduction was similar in magnitude to the CBF reductions observed in previous studies in which nonspecific NOS inhibitors were used. In the present study, 7-NI did not increase the MABP. Furthermore, the CBF response to oxotremorine, a blood–brain barrier permeant muscarinic agonist that induces cerebrovasodilation via endothelium-derived NO, was unaffected by 7-NI. These results confirmed that 7-NI does not influence eNOS; they also indicated that the effects of 7-NI on the resting CBF and on the CBF response to hypercapnia in this study were solely related to its inhibitory action on nNOS. The results further suggest that the NO synthesized by the action of nNOS participates in regulation of basal CBF and is the major, if not the only, category of NO contributing to the hypercapnic CBF response.

Effects of Estrogen on Leukocyte Adhesion After Transient Forebrain Ischemia

Background and Purpose Recent findings indicate that estrogen (ie, 17&bgr;-estradiol [E2]) 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 E2-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 E2-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 E2-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.

Role of nitric oxide, adenosine, N-methyl-D-aspartate receptors, and neuronal activation in hypoxia-induced pial arteriolar dilation in rats.

In this study, we tested the hypothesis that nitric oxide (NO) and adenosine (ADO) are the principal mediators of severe hypoxia-induced vasodilation. In addition, we examined whether activation of N-methyl-D-aspartate (NMDA) receptors and/or perivascular nerves plays a role. A closed cranial window and intravital microscopy system was used to monitor diameter changes in pial arterioles (approximately 40 microns) in anesthetized rats. The relative contributions of ADO, NMDA, NO, and neuronal activation to hypoxic cerebrovasodilation were assessed using the blockers 8-sulfophenyltheophylline (8-SPT), MK-801, nitro-L-arginine methylester (L-NAME), and tetrodotoxin (TTX). Two experimental series were studied. In the first, we tested the effects of NOS inhibition, via topical L-NAME (1 mM), on moderate (PaO2 approximately 46 mmHg) then severe (PaO2 approximately 34 mmHg) hypoxia-induced dilation. To confirm that L-NAME was affecting specifically NO-dependent responses, we also examined, in each experiment, the vasodilatory responses to topical applications of NOS-dependent (adenosine diphosphate (ADP); acetylcholine (ACh)) and -independent (sodium nitroprusside (SNP)) agents, in the presence of L-NAME or, in controls, the presence of D-NAME or no added analogue. In the second series, topical suffusions of ADP, ADO, and NMDA were sequentially applied, followed by 5 min exposure to severe hypoxia (PaO2 approximately 32 mmHg). Following return to normoxia, a suffusion of either 8-SPT (10 microM), MK-801 (10 microM), TTX (1 microM), or 8-SPT+MK-801 was initiated (or, in controls, application of a drug-free suffusate was maintained), and the above sequence repeated. In control, TTX, and 8-SPT+MK-801 experiments, baseline conditions were then restored and hypercapnia (PaCO2 = 70-85 mmHg) was imposed. In the series 1 control groups, moderate and severe hypoxia elicited approximately 20% and 35-40% increases in diameter, respectively. L-NAME attenuated ADP- and ACh-induced dilations, did not alter the arteriolar responses to SNP or moderate hypoxia, but prevented further dilation upon imposition of severe hypoxia. This suggested that 45-50% of the severe hypoxia response was NO-dependent. In series 2, 8-SPT blocked the adenosine response and reduced severe hypoxia-induced dilation by 46%. MK-801 predictably blocked NMDA-induced relaxation and reduced the hypoxic response by 42%. When combined, 8-SPT and MK-801 affected hypoxic vasodilation additively. After TTX, the ADP and ADO responses were normal, but NMDA and hypoxia responses were completely blocked. Hypercapnia-induced dilation was unaffected by TTX or 8-SPT+MK-801. The results imply that severe hypoxia-induced release of NO and ADO, and the accompanying pial arteriolar dilation, are wholly dependent on the capacity to generate action potentials in perivascular nerves. The similarity of the L-NAME and MK-801 effects on hypoxic cerebrovasodilation suggests that the NO-dependency, to a large degree, derives from NMDA receptor activation.

The Role of Endothelium and Nitric Oxide in Rat Pial Arteriolar Dilatory Responses to CO2 in vivo

Using a closed cranial window system and intravital microscopy/videometry, we studied the rat pial arteriolar (30–60 μm) responses to CO2 before and following a light/dye (L/D) endothelial injury or topical application of the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine (L-NA) or its inactive form, D-NA. L/D treatment consisted of intravenous injection of sodium fluorescein and the illumination (for 90 s) of arteriolar discrete segments on the cortical surface with light from a mercury lamp. Functional changes in pial arteriolar endothelium were characterized by evaluating responses to topical application of acetylcholine (Ach, 5 × 10−4 M) and to intravenous (i.v.) oxotremorine (OXO, a stable blood–brain barrier permeant muscarinic agonist, 1 μg kg−1 min−1). After the L/D injury, dilation to Ach was absent whereas dilations to the NO donor, S-nitrosoacetyl-penicillamine (SNAP, 10−5 M) and to CO2 (5%) were unchanged (Paco2 = 70 mm Hg). Loss of Ach response but intact SNAP response confirmed functional endothelial injury and intact smooth-muscle function. The global endothelium-dependent vasodilation induced by i.v. OXO was markedly attenuated when expanding the L/D injury field from 300 μm to 6 mm in diameter. However, the global vasodilation induced by inhalation of CO2 was still unaffected by this increase in the area of light exposure. This provides evidence that the expanded exposure was capable of impairing global vasodilation resulting from endothelium-dependent stimuli but not from inhalation of CO2. The intact CO2 response despite an endothelial dysfunction suggests that the reported NO dependence of hypercapnia-induced cerebral hyperemia in rats cannot be attributed to an endothelial NO source. Topical suffusion of L-NA (1 mM) for 45–60 min in our preparation blocked the pial arteriolar response to Ach, whereas CO2 and SNAP responses were unaffected. An attenuation (by 50%) of the response to CO2 was achieved if suffusion of L-NA was given for ≥2 h. Suffusion of D-NA, applied in the same manner, did not influence responses to any of the above applications. This demonstrates that there is a NO-dependent component for hypercapnic cerebral vasodilation even at the pial arteriolar level. The strikingly different time-related effect of topical L-NA on the Ach and CO2 responses, together with the lack of effect of endothelial injury on CO2-induced dilation, strongly suggest a nonendothelial source of NO in hypercapnic cerebrovascular dilation.

Halothane Vasodilation and Nitric Oxide in Rat Pial Vessels

We investigated whether halothane (HAL), administered via cerebral cortical suffusion at concentrations of 1, 2, and 3%, could induce cerebral microvascular dilatation in vivo and whether the vasodilatory response was dependent on nitric oxide (NO) synthesis. The studies were performed using N2O/fentanyl-anesthetized, paralyzed, and mechanically ventilated rats. A closed cranial window and an intravital microscopy technique were employed. This system permitted the controlled delivery of various vasoactive agents in an artificial cerebrospinal fluid (aCSF) solution and the measurement of diameters of pial arterioles and venules. Each experiment included evaluations of (a) the direct smooth muscle relaxing action of NO, using sodium nitroprusside (SNP), and (b) the capacity for generation and release of endogenous NO, using adenosine diphosphate (ADP). Following confirmation of an intact NO-relaxing and generating capacity, HAL (in aCSF) was suffused at increasing concentrations. Nitric oxide synthase (NOS) inhibition was established with topical nitro-L-arginine (L-NA) or its methyl ester (L-NAME) and the above sequence repeated. The results for rats treated with L-NA (n = 5) or L-NAME (n = 5) were analyzed separately and as a combined group. No significant differences in vascular responses were observed when comparing the two groups. Initially, both SNP and ADP produced significant diameter increases (all groupings) in arterioles (14-28% change) and venules (14-25% change). For all groups, suffusions of 1 to 3% HAL produced arteriolar dilation, ranging from a 10 to 25% increase over baseline diameter. A statistically significant dose dependency was only observed with the combined data.(ABSTRACT TRUNCATED AT 250 WORDS)

Response entropy increases during painful stimulation.

Frontal electromyography (FEMG) may increase during painful stimulation and indicate patient arousal. The Datex-Ohmeda Entropy Module calculates state entropy (SE) of the electroencephalogram (EEG; 0.8-32 Hz) and response entropy (RE) of EEG and FEMG (0.8-47 Hz). We determined whether RE increases above SE (RE − SE), an indication of FEMG, increase during painful stimuli and if this is related to paralysis or level of anesthesia. With the unanesthetized baseline measurement, SE was 89 ± 2 and RE was 98 ± 2. During paralysis and anesthesia with either 0.8% (n = 10) or 1.4% (n = 10) isoflurane, SE decreased to 63 ± 7 and 34 ± 14, respectively, and the RE − SE difference decreased 90%. Before recovery from paralysis, arterial catheter or head pin placement increased RE − SE above unanesthetized levels in eight patients (five treated with 0.8% and three with 1.4% isoflurane), consistent with an increase in FEMG. The elevated RE − SE difference was related to a significant increase in SE, blood pressure, and heart rate. After recovery from paralysis, tetanic stimulation of the ulnar nerve increased the RE − SE difference above unanesthetized levels in 8 of 20 patients (6 treated with 0.8% and 2 with 1.4% isoflurane). In these patients, SE increased significantly. The remaining 12 patients did not show an increase in RE − SE during tetanic stimulation and SE did not increase. We conclude that increased RE during painful stimulation was not dependent on recovery from paralysis but was seen more often in patients anesthetized with 0.8% compared with 1.4% isoflurane. This suggests that RE reflects FEMG and may be useful to identify inadequate anesthesia and patient arousal during painful stimuli.

Estrogen inhibits NF??B-dependent inflammationin brain endothelium without interfering withI??B degradation

The protective effects of 17beta-estradiol in cerebral ischemia may be partially due to the blockade of leukocyte adhesion in cerebral endothelial cells, although the molecular mechanisms are not well understood. We report that 17beta-estradiol (E(2)), but not the alpha-enantiomer, inhibited the basal and interleukin-1beta (IL-1beta)-mediated expression of the intercellular adhesion molecule type 1 (ICAM1) and NFkappaB activation, in cultured brain endothelial cells. However, the degradation of IkappaB-alpha, which is an essential requirement for the translocation of NFkappaB to the nucleus, and a common biological target to suppress NFkappaB activation, was not halted by E(2). These findings indicate that decreased expression of adhesion molecules may account for the capacity E(2) to reduce adhesion of leukocytes in cerebral endothelium in vivo, and suggest the existence of brain-specific, estrogen-sensitive pathways, other than IkappaB-alpha_-regulation, to modulate NFkappaB. The stereoselectivity of the E(2) effect is consistent with an estrogen receptor-mediated mechanism.

Protein kinase C suppresses receptor-mediated pial arteriolar relaxation in the diabetic rat.

Cerebral vasodilatory responses are selectively impaired in chronically hyperglycemic, diabetic rats. In this study, we tested the hypothesis that chronic hyperglycemia-induced protein kinase C (PKC) activation can account for the suppression of 2 separate receptor-mediated vascular relaxation processes: (1) endothelium-derived nitric oxide (NO) release, and (2) NO-independent beta-adrenergic receptor (beta-AR) activation. The in vivo reactivity of pial arterioles was evaluated in anesthetized rats (streptozotocin-treated diabetics and controls) using a closed cranial window and intravital microscopy. Compared with controls, diabetic rats showed a substantial attenuation or loss of the arteriolar relaxation response accompanying suffusion of the receptor-linked, NO-dependent agonists, acetylcholine (Ach) and adenosine diphosphate (ADP), and the beta-AR-agonist, isoproterenol (ISO). The vasodilatation induced by the direct NO donor, sodium nitroprusside (SNP), was the same in both groups. In the presence of the PKC inhibitor, staurosporine (STAURO), the Ach, ADP, and ISO responses were, largely restored and the SNP response was unaffected. STAURO produced no changes in Ach, ADP, ISO, or SNP responses in non-diabetic rats. These results suggest that PKC activation in chronically hyperglycemic, diabetic rats suppresses receptor-dependent NO release and desensitizes beta-ARs.

Role of nitric oxide and endothelium in rat pial vessel dilation response to isoflurane

Isoflurane induces cerebral hyperemia. We sought to assess whether isoflurane induces cerebral microvessel dilation in vivo, and if so, to determine whether nitric oxide (NO) and endothelium are involved. By using a rat closed cranial window model, pial arterioles and venules of 30-70 microns in diameter were measured using intravital microscopy. The cerebral microvascular dilatory response was recorded as percent change of diameter from baseline. The pial vessels were suffused with sodium nitroprusside (SNP) or S-nitroso-acetyl-penicillamine (SNAP) to verify intact vascular smooth muscle relaxation function, and with adenosine diphosphate (ADP) and/or acetylcholine (ACh) to verify endothelial NO-generating capability. To isolate NOs role in the cerebral microvascular effects of isoflurane (Protocol I), microvessels were studied with and without nitric oxide synthase (NOS) inhibition by topically applied nitro-L-arginine methyl ester (L-NAME). In controls, L-NAME was replaced by its inactive enantiomer, nitro-D-arginine methyl ester (D-NAME). Mercury light plus fluorescein dye (LD) endothelial injury (Protocol II) was used to delineate an endothelium-mediated mechanism. Subsequently, vasodilator applications were repeated to verify the desired effects of the interventions and followed by suffusion of isoflurane 1%, 2%, and 3% (Protocol I) or isoflurane 3% (Protocol II). Suffusions of SNP, ADP, and ACh induced diameter increases of 15%-30%. NOS inhibition with L-NAME greatly attenuated ADP and ACh responses, but did not alter the SNP response, confirming that NO generation was blocked, but not NO action. These responses were unaffected in D-NAME-suffused rats. Isoflurane dilated arterioles 17% and venules 6% in the presence of D-NAME suffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of recombinant human erythropoietin in a Jehovah's Witness

We report the case of a Jehovahs Witness who bled massively, refused blood transfusion, and survived profound anemia (hematocrit = 5.6%) intact. The patient was treated with recombinant human erythropoietin, parenteral iron, and oxygen. The pharmacology and hematopoietic response to erythropoietin are discussed. We suggest considering this therapy for acutely anemic patients who refuse transfusion to decrease the duration of the most severe anemia.