Background and PurposeWe previously reported that L-arginine infusion increased pial vessel diameter by nitric oxidedependent mechanisms, improved regional cerebral blood flow (rCBF) distal to middle cerebral artery (MCA) occlusion, and reduced infarction volume in spontaneously hypertensive rats when administered intraperitoneally before and after MCA occlusion. In this report we extend our findings (1) by examining the time course of L-arginine on rCBF and pial vessel diameter under basal conditions and on rCBF after MCA occlusion and (2) by reproducing the protective effect of L-arginine on infarct volume when given intravenously immediately after the onset of MCA occlusion in both normotensive and hypertensive models of focal cerebral ischemia.Methods Changes in pial vessel diameter (closed cranial window) and rCBF (laser-Doppler flowmetry) were measured over time after L-arginine infusion into anesthetized Sprague-Dawley rats. rCBF was also measured distal to MCA occlusion in a brain region showing rCBF reductions in the range of 80% of baseline. The effects of infusing L-arginine (300 mg/kg for 10 minutes beginning 5 minutes after occlusion) were assessed on infarction volume in Sprague-Dawley rats after proximal MCA occlusion and in spontaneously hypertensive rats after common carotid artery plus distal MCA occlusion.

Summary:We examined whether 7-nitroindazole (7-NI), a putative inhibitor of neuronal nitric oxide synthase (nNOS), decreases cerebral infarction 24 h after proximal middle cerebral artery (MCA) occlusion. In preliminary experiments, we determined that 7-NI (25, 50, and 100 mg/kg i.p.) decreased nitric oxide synthase (NOS) activ ity within cerebral cortex by 40-60% when measured up to 120 min, but not 240 min after administration. At 25 or 50 mg/kg, 7-NI did not alter the systemic arterial blood pressure or the dilation of pial arterioles after topical ace tylcholine (10 and 100 j-LM). To examine the effect of 7-NI on infarct size, 55 Sprague-Dawley halothane anesthetized rats were subjected to proximal MCA occlu sion (modified Tamura method). Five minutes after oc clusion, 7-NI (25 or 50 mg/kg i.p.) or vehicle was injected. Animals treated with 25 or 50 mg/kg showed 25 and 27%

The authors studied the venous drainage system and its impairment in relation to risk of hemorrhage in 108 cases of supratentorial arteriovenous malformation (AVM). The proportion of AVM's undergoing hemorrhage (hemorrhagic rate) was calculated in relation to: 1) the number of draining veins (one, two, or three or more); 2) the presence or absence of impairment in venous drainage (severe stenosis or occlusion in draining veins); and 3) the location of draining veins (deep venous drainage alone, superficial venous drainage alone, or a combination of the two). Statistical analysis demonstrated that AVM's with the following characteristics had a high risk of hemorrhage: 1) one draining vein (hemorrhagic rate 89% in 54 patients); 2) severely impaired venous drainage (hemorrhagic rate 94% in 18 patients); and 3) deep venous drainage alone (hemorrhagic rate 94% in 32 patients). The present study suggests that the venous drainage system of AVM's is significantly associated with the risk of hemorrhage of these lesions. Therefore, careful preoperative angiographic evaluation of the venous drainage system is mandatory for decision making in the management of patients with AVM's.

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Summary: Control of physiological parameters such as respiration, blood pressure, and arterial blood gases has been difficult in the mouse due to the lack of technology required to monitor these parameters in small animals. Here we report that anesthetized and artificially venti lated mice can be maintained under physiological control for several hours with apparently normal cerebrovascular reactivity to hypercapnia and mechanical vibrissal stim ulation. SV -129 mice were anesthetized with urethane (750 mg/kg i.p.) and a-chloralose (50 mg/kg i.p.), intu bated, paralyzed, and artificially ventilated. Respiratory control was maintained within physiological range by re ducing the inspiratory phase of the respiratory cycle to <0. 1 s and by adjusting end-tidal CO2 to give a Pe02 of 35 ± 3 mm Hg. In these mice, mean arterial pressure (95 ± 9 mm Hg), heart rate (545 ± 78 beats/min), and arterial pH (7.27 ± 0. 10) could be maintained for several hours.Recent advances in transgenic technology and ge netic engineering have emphasized the need to de velop accurate methods for monitoring and control ling respiration and systemic arterial blood pressure in the anesthetized laboratory mouse (Aguzzi et aI. , 1994). Unlike larger animals that can sustain fre quent blood samplings and volume depletion, sys temic hypotension develops in the mouse after withdrawing only a few hundred microliters of blood. Even catheterizing blood vessels can be technically demanding. Ventilatory control poses the greatest challenge, however, because most small animal ventilators simply divide the respira-

Summary:We assessed the regional cerebral blood flow (rCBF) response to vibrissal stimulation before and after nitric oxide synthase (NOS) inhibitors were topically ap plied through a closed cranial window placed over the cortical barrel fields in anesthetized Sprague-Dawley rats. In the presence of L-nitroarginine (l mM), both the maximum and total responses became reduced, but only in those animals demonstrating >50% inhibition of NOS activity as determined by the conversion of [ 3 H]arginine to eHlcitrulline within homogenates taken from cortical gray matter under the cranial window. The degree of en-The mechanism which couples blood flow and metabolism within the brain is not well understood. Recently, nitric oxide was suggested as a potential mediator because it is formed and released from neurons (Garthwaite et aI., 1988; Knowles et aI., 1989; Bredt et aI., 1990), diffuses readily (e.g., 0.5 fJ.m theoretical distance) (Knowles and Moncada, 1992), crosses cellular membranes freely, and is a potent vasodilator (Palmer et al., 1987). Although certain publications note that nitric oxide synthase (NOS) inhibitors attenuate the blood flow response elicited by somatosensory stimulation (Northington et aI., 1992; Dirnagl et aI., 1993), others have not observed this relation (Adachi et aI., 1993; N gai et aI., 1993).Studies were undertaken to establish a possibleReceived June 18 , 1993; final revision received July 6, 1993; accepted July 6, 1993.Address correspondence and reprint requests to Dr. Michael A. Moskowitz, Massachusetts General Hospital, 32 Fruit Street, Boston, MA 02114, U.S.A.Abbreviations used: CSF, cerebrospinal fluid; EDTA, ethyl enediaminetetra-acetic acid; HEPES, N-2-hydroxyethylpipera zine-N' -2-ethanesulfonic acid; L-NA, NG-nitro-L-arginine; L-NAME, �-nitro-L-arginine methylester; �-NADPH, �-nico tinamide adenine dinucleotide phosphate; NOS, nitric oxide syn thase; rCBF, regional CBF. 45zyme inhibition depended in part, upon duration after topical application of NOS inhibitor. When >50%, en zyme inhibition correlated with the decrease in maximum and total rCBF response (p < 0.01). These findings em phasize the merits of assessing enzyme activity after ad ministering NOS inhibitors, and suggest that NO gener ated from parenchymal NOS activity plays an important role in the cerebrovascular response to physiologic so matosensory stimulation under the stated conditions. Key Words: Nitric oxide-Nitric oxide synthase-L Nitroarginine-Cortical barrel fields-rCBF.role for NO as a coupling mediator for regional CBF (rCBF) and metabolism, and to extend our own pre liminary negative findings examining the effects of topical NOS inhibitors on the rCBF response to vibrissal stimulation 30 min after topical application (Irikura et aI., 1993). Accordingly, NOS activity was determined in vitro based on the conversion of eH]arginine to [3H]citrulline within homogenates taken from subjacent cerebral cortex. Our results support an important role for NO under the condi tions of this assay, and for the need to asse...

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Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, NO-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by Nwnitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO. Nitric oxide (NO) appears to mediate the cerebral blood flow increase during moderate hypercapnia (5% CO2) (1). Inhibitors of NO synthase (NOS, EC 1.14.13.39) activity such as Nw-nitro-L-arginine (L-NNA) or Nw-nitro-L-arginine methyl ester (L-NAME) attenuate the regional cerebral blood flow (rCBF) increase during CO2 inhalation in most studies (2-7), and the attenuated response correlates with inhibition of brain NOS activity as measured ex vivo by the conversion of [3H]arginine to [3H]citrulline (3). NO may act via cGMP, as 8-bromo-cGMP as well as an NO donor restores the hypercapnic response after treatment with NOS inhibitors (8).At least three NO sources may contribute to the CO2-induced blood flow response-the endothelium, perivascular nerves, and parenchymal neurons (1). The relative contributions of these sources have been difficult to establish because L-NNA and L-NAME are nonselective, inhibiting neuronal NOS (nNOS) and endothelial NOS (eNOS) similarly (9).A mutant mouse strain (Kn) lacking expression of nNOS has been developed (10). Residual brain NOS activity, <5% of wild type, may derive from eNOS which is expressed in brains of the Kn mutants in neurons as well as blood vessels (11). We recently established a physiological monitoring system which permits continuous monitoring of arterial blood pressure, end-tidal CO2, rCBF (laser Doppler flowmetry), heart rate, and pial vessel diameter in the artificially ventilated mouse (12, 13). We now show that nNOS is a mediator of C02-dependent hyperemia in wild-type mice. Kn mice display normal CO2 responsiveness which is achieved by non-NO, cGMP-independent mechanisms, reflecting true physiological redundancy. METHODSImmunohistochemistry. Mice were perfused with phosphate-buffered saline followed by freshly depolymerized 4%The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 2-4 hr. Immunostaining for eNOS and nNOS was performed as described (14) with affinity-purified eNOS antiserum (1:50 d...