Background and Purpose-Recent findings indicate that estrogen (ie, 17␤-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

Summary:The nitric oxide synthase (NOS) inhibitors, ni tro-L-arginine, its methyl ester, and N-monomethyl-L arginine, have been shown to attenuate resting CBP and hypercapnia-induced cerebrovasodilation. Those agents nonspecific ally 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 CBP during normocapnia and hypercap nia in fentanyUN20-anesthetized rats. CBP was moni tored using laser-Doppler flowmetry. Administration of 7-NI (80 mg kg-I i.p.) reduced cortical brain NOS activ ity by 57%, the resting CBP by 19-27%, and the CBP response to hypercapnia by 60%. The 60% reduction was similar in magnitude to the CBP reductions observed in previous studies in which nonspecific NOS inhibitors Nitric oxide (NO) has emerged as an important endogenous modulator of brain function, and has been shown to play a role in the cerebral vasodila tory responses (CVDRs) to a wide variety of stim uli, including, muscarinic agonists, neuronal activa tion, volatile anesthetics, hypoxia, and hypercapnia (Iadecola et aI., 1994). NO is produced by the action of NO synthase (NOS) (Luscher and Vanhoutte, 1990). In the brain, NOS is not only located in the Received June 2, 1994; final revision received December 16, 1994; accepted December 20, 1994. Address correspondence and reprint requests to Dr. Q. Wang at Department of Anesthesiology, Michael Reese Hospital, 2929 S. Ellis Ave., Chicago, IL 60616, U. S.A.Abbreviations used: AMB, atropine methyl bromide; CVDRs, cerebral vasodilatory responses; EDT A, ethylenediamine tetraacetic acid; HEPES, N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid; L-NA, nitro-L-arginine; L-NAME, methyl ester of L-NA; L-NMMA, N-monomethyl-L-arginine; 7-NI, 7-nitroindazole; NO, nitric oxide; NOS, NO synthase; eNOS, constitutive NOS; eNOS, endothelial NOS; iNOS, inducible NOS; nNOS, neuronal NOS; OXO, oxotremorine. 774were used. In the pre�ent study, 7-NI did not increase the MABP. ,Furthermore, the CBP response to oxotremo rine, 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 CBP and on the CBP response to hypercapnia in this study were solely related to its inhibitory action on nNOS. The results fur ther suggest that the NO synthesized by the action of nNOS participates in regulation of basal CBP and is the major, if not the only, category of NO contributing to the hypercapnic CBP response. Key Words: Hypercapnia-7 -NI-N itric oxide-eN OS-nN OS-Oxotremorine.endothelium, but also in neurons, perivascular nerves, and astrocytes (Bredt et aI., 1990; Murphy et aI., 1993). There are at least two constitutive NOS (cNOS) isoforms in the brain (i.e., the endo thelial [eNOS] and neuronal [nNOS] forms) that generate NO phasically (Moncada et aI., 1991). Ow ing to the rapidity of the CVDRs to the abo...

Summary: The role of nitric oxide (NO) synthesis in the cerebral hyperemic responses to hypercapnia and hypox ia was investigated in anesthetized rats. Regional CBF (rCBF) measurements were obtained in the cortex (CX), subcortex (SC), brain stem (BS), and cerebellum (CE) us ing radiolabeled microspheres. The rCBF responses to either hypercapnia (P ac02 = 70-80 mm Hg) or hypoxia (P a02 = 40--4 5 mm Hg) were compared in rat groups stud ied in the presence and absence of NO synthase inhibition induced via the intravenous infusion of nitro-L-arginine methyl ester (L-NAME, 3 mg kg-1 min-I). Administra tion of L-NAME under normocapnic/normoxic conditions produced a 40-60% reduction in baseline rCBF values, indicating the presence of a NO "tone" in the cerebral vasculature. Infusion of L-NAME resulted in a substan tial attenuation, in all regions measured, of the rCBF in creases that normally accompany hypercapnia. In com paring saline-infused to L-NAME-infused rats, the per centage increases in rCBF (from normocapnic baseline values) were 351% versus 166% (CX), 446% versus 199% (SC), 443% versus 206% (BS), and 483% versus 174% (CE), respectively. The rCBF changes from baseline Vascular tissue, when exposed to certain physi ologic stimuli (e,g., activation of specific receptors, shear stress), can exhibit an endothelium dependent vasodilation in a wide range of struc tures, including the brain, via the synthesis and re lease of nitric oxide (NO) or nitrosothiol com pounds (Luscher and Vanhoutte, 1990; Myers et Received February 11, 1992; Abbreviations used: BS, brainstem; CE, cerebellum; CYR, cerebral vascular resistance; CX, cortex; EAA, excitatory amino acid; EDRF, endothelium derived relaxing factor; L-NAME, ni tro-L-arginine methyl ester; MANOY A, multivariate analysis of variance; NOS, nitric oxide synthase; PKC, protein kinase C; SC, subcortex. 80(�rCBF in ml 100 g -I min -I ) were 488 versus 57 (CX), 570 versus 60 (SC), 434 versus 72 (BS), and 393 versus 45 (CE), respectively. These differences were all statistically significant (p < 0.05). During hypoxia, when compared to rats not given L-NAME, inhibition of NO synthase activ ity resulted in significantly greater (p < 0.05) percentage increases in rCBF (from normoxic baseline values) in most regions. The changes in non-L-NAME-vs.L-NAME-infused rats were 156% versus 262% (CX), 181% versus 309% (SC), and 210% versus 462% (BS), respectively. When the �rCBF values (from normoxic baseline levels) were compared, the changes were greater in the L-NAME group, but the differences were statisti cally insignificant. The results of this study indicated that NO synthesis is critically involved in the cerebral hy peremic response to hypercapnia but not hypoxia. In fact, the data obtained in the hypoxic groups suggested that reductions in O2 supply may inhibit the NO-generating capacity in the brain. Key Words: Cerebral blood flow Endothelium-dependent relaxation-Hypercapnia Hypoxia-L-NAME-Nitric oxide-Nitric oxide syn thase.

We examined whether damage to the glia limitans (GL), via exposure to the gliotoxin L-␣-aminoadipic acid (L-␣AAA), alters hypercapniainduced pial arteriolar dilation in vivo. Anesthetized female rats were prepared with closed cranial windows. Pial arteriolar diameters were measured using intravital microscopy. L-␣AAA (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-␣AAA 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 -nitro-L-arginine (L-NNA). Subsequent blockade of prostanoid synthesis, via indomethacin (Indo), reduced CO2 reactivity to 10 -15% of normal. In vehicle-treated controls, L-NNA, followed by Indo, reduced the response to ϳ50% and then to 15-20% of the normocapnic value, respectively. On the other hand, L-␣AAA 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-␣AAA actions on pial arterioles or influence on neuronal transmission. Furthermore, L-␣AAA 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 hypercapniaversus 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 ϳ65%) 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. connexin; gap junction; hypercapnia; L-␣-aminoadipic acid; nitric oxide; prostanoids BECAUSE OF THE INTIMATE ANATOMIC RELATIONSHIP between cerebral resistance vessels and astrocytes (19), it has been postulated that astrocytes play a vital role in the regulation of local perfusion in the brain (7). In fact, experimental evidence to that effect has been published (43). Small pial arterioles (Ͻ50 m), although exhibiting a relative paucity of direct neural connections (8, 33), nevertheless appear to be regulated by extravascular influences (23,38). F...

Summary: Using a closed cranial window system and in travital microscopy/videometry, we studied the rat pial arteriolar (30-60 !Lm) responses to CO2 before and fol lowing a light/dye (LID) endothelial injury or topical ap plication of the nitric oxide synthase (NOS) inhibitor, ni tro-L-arginine (L-NA) or its inactive form, D-NA. LID 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 en dothelium were characterized by evaluating responses to topical application of acetylcholine (Ach, 5 x 10-4 M) and to intravenous (i.v.) oxotremorine (OXO, a stable blood-brain barrier permeant muscarinic agonist, 1 !Lg kg -1 min -1 ) . After the LID injury, dilation to Ach was absent whereas dilations to the NO donor, S-nitroso acetyl-penicillamine (SNAP, 10-5 M) and to CO2 (5%) were unchanged (Paco2 = 70 mm Hg). Loss of Ach re sponse 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 LID injury field from 300 !Lm to 6 mm in diameter. However, the global vasodilation induced by inhalation ofNitric oxide (NO) is a rapidly diffusible and po tent dilator of vascular smooth muscle (Moncada et ai., 1991). It is synthesized from L-arginine, in com bination with molecular oxygen, by nitric oxide synthase (NOS) (e.g., Luscher and Vanhoutte, 1990), a calcium/calmodulin and NADPH-depen- Abbreviations used: Ach, acetylcholine; aCSF, artificial CSF; ICP, intracranial pressure; eNOS, endothelial nitric oxide syn thase; iNOS, inducible nitric oxide synthase; LID, light/dye; L-NA, nitro-L-arginine; NOS, nitric oxide synthase; OXO, oxo tremorine; SNAP, S-nitr.oso-acetyl-penicillamine; TTX, tetrodo toxin. 944CO2 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 re SUlting from endothelium-dependent stimuli but not from inhalation of CO2, The intact CO2 response despite an endothelial dysfunction suggests that the reported NO de pendence 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 CO 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 re sponses to any of the above applications. This demon strates that there is a NO-dependent component for hy percapnic cerebral vasodilation even at the pial arteriolar level. The strikingly different time-related effect of topi cal L-NA on the Ach and CO2 responses, together with the lack of effect of endothelial injury on CO2-induced dilation, strongly sugg...

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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.

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.