Nitric oxide participates in the cerebrovasodilation elicited from cerebellar fastigial nucleus COSTANTINO Department
IADECOLA of Neurology,
University
of Minnesota
Iadecola, Costantino. Nitric oxide participates in the cerebrovasodilation elicited from cerebellar fastigial nucleus. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1156R1161, 1992.-The endothelium-derived relaxing factor, probably NO, is a potent vasodilator that mediates the vasodilating action of acetylcholine (ACh). We studied whether NO participates in the cholinergic cerebrovasodilation elicited by stimulation of the cerebellar fastigial nucleus (FN). Rats were anesthetized with halothane and ventilated. FN or pontine reticular formation (PRF) were stimulated through microelectrodes. Hypertension was prevented by spinal cord transection with arterial pressure maintained by intravenous phenylephrine. Cerebral blood flow (CBF) was continuously monitored through a cranial window over the sensory cortex by a laser-Doppler probe. The window was superfused with Ringer solution (pH 7.3-7.4; 37°C). During Ringer superfusion FN stimulation (100 ,uA; 50 Hz) increased CBF by 90 t 7% (n = 27; P < 0.001, analysis of variance and Tukey’s test) and PRF stimulation (100 PA; 100 Hz) by 128 t 18% (P < 0.001; n = 9). Superfusion with the guanylyl cyclase inhibitor methylene blue (MB) (1 mM) attenuated the CBF increase elicited by FN stimulation by 77 t 3% (n = 22; P < 0.001). MB did not affect the CBF increase elicited by PRF stimulation (+98 t 18%; n = 9; P > 0.05). Similarly, superfusion with the NO-synthase inhibitor nitro-L-arginine (L-NA) attenuated the CBF increase elicited by FN stimulation (-67 t 3%; n = 14; P < 0.001 from Ringer) but not PRF stimulation (P > 0.05; n = 9). The CBF increases elicited by FN stimulation were not affected by the inactive isomer of nitroarginine, D-NA (P > 0.05; n = 7). Thus the neocortical vasodilation elicited by FN stimulation is substantially attenuated by inhibition of NO synthesis or inhibition of the enzyme mediating NO action. The results indicate that the vasodilation elicited by FN stimulation is mediated, for the most part, by arginine-derived NO and suggest that NO may be important also in the regulation of the cerebral circulation. cerebellum; cerebral blood flow; methylene blue; nitroarginine; endothelium-derived relaxing factor STIMULATION of the cerebellar fastigial nucleus (FN) increases cerebral blood flow (CBF) globally through neural pathways entirely contained within the brain (13, 15, 24, 26). The increases in CBF are greatest in regions of the cerebral cortex, wherein the vasodilation is not associated with increased glucose utilization (23). In neocortex, the increase in CBF is substantially attenuated by topical application of atropine (I), a finding indicating that the vasodilation is mediated by local release of acetylcholine (ACh). While it is well established that ACh is a powerful cerebrovascular vasodilator (lo), the local mechanisms by which ACh produces vascular relaxation during FN stimulation remain to be elucidated. There is increasing evidence that the vascular relaxation elicited by ACh is mediated by an endothelium-derived relaxing factor (EDRF) (5, 9, 20, 27), a labile substance that is probably nitric oxide (NO) or a closely related nitroso compound (2 I). It is therefore conceivable that during FN stimulation ACh mediates the cerebrovasodilation through NO. ELECTRICAL
R1156
0363-6119/92
$2.00
Copyright
Medical
School, Minneapolis,
Minnesota
55455
In the present study we sought to determine whether NO participates in the cerebrovasodilation elicited by FN stimulation. NO is synthesized from L-arginine by the enzyme NO-synthase (3, 25, 29), whose activity can be inhibited by N”-substituted arginine analogues such as nitroarginine (L-NA) (19, 21, 29). Furthermore, NO exerts its vasodilating action by activating soluble guanylyl cyclase and increasing 3’,5’-cyclic guanosine monophosphate (cGMP) production in smooth muscles (18). We therefore studied whether inhibition of NO synthesis by NA or inhibition of NO action by the guanylyl cyclase inhibitor methylene blue (MB) attenuates the increase in CBF elicited by FN stimulation. We shall demonstrate that topical superfusion of these agents on the parietal cortex substantially attenuates the local cerebrovasodilation elicited by FN stimulation while the increases in CBF elicited by stimulation of the pontine reticular formation (PRF) are not affected. METHODS Methods for surgical preparation of rats, monitoring of CBF by laser-Doppler flowmetry (LDF), and brain stimulation have been described in detail previously (12-15, 17, 24) and will be summarized below. General procedures. Studies were performed on 25 male Sprague-Dawley rats weighing 290-380 g. Rats were anesthetized with 5% halothane in 100% oxygen, delivered through a facial mask. After induction of anesthesia, halothane was administered at a reduced rate (2%). Catheters were inserted in both femoral arteries and in the right femoral vein, and the trachea was cannulated. Animals were then placed on a stereotaxic frame (Kopf) and artificially ventilated with 100% oxygen by a mechanical ventilator (rodent respirator, Harvard Apparatus). Body temperature was maintained at 37 t 0.5”C using a heating lamp thermostatically controlled by a rectal probe (YSI). One of the arterial catheters was used for continuous recording of arterial pressure (AP), mean AP, and heart rate on a chart recorder (Grass), while the other arterial line was used for blood sampling. The venous catheter was used for intravenous administration of phenylephrine after transection of the spinal cord (see below). Arterial Pco~, PO,, and pH were measured at multiple times on 100 ~1 of blood using a blood gas analyzer (Corning 178). Spinal cord transection. To prevent the effect on CBF of the hypertension elicited by FN or PRF stimulation, the spinal cord was transected at the first cervical segment. As described elsewhere (17, 24), the spinal cord was exposed at the C,-C, junction by removing the posterior lamina of the atlas. After removing the dura, the cord was severed at C1, and a small segment below C, was removed by suction. Just before the procedure, an intravenous infusion of phenylephrine (1.3-6.4 pg/min) was started to counteract the fall in AP that occurs after spinal cord transection. The completeness of the transection was verified at autopsy. Monitoring of CBF by laser-Doppler flowmetry. LDF was performed using a Vasamedic flowmeter (TSI, St. Paul, MN) (12, 14). A 3 x 3 mm cranial window was drilled at a site 2-3 mm lateral and 1-2 mm caudal to bregma. The underlying cortex
0 1992 the American
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corresponds to the leg area of the primary sensory cortex (14). The dura was carefully removed and the craniotomy site was continuously superfused at a rate of 0.33 ml/min with Ringer warmed at 37°C and aerated with 95% 0, and 5% CO, (pH 7.3-7.4). The composition of the Ringer (in mM) was 143.5 Na+, 3.0 K+, 1.5 Ca2+, 1.4 Mg2+, 115 Cl-, 26.4 HCO;, 9.6 gluconate, and 5.0 glucose (14). The LDF probe (tip diameter 0.8 mm) was mounted on a micromanipulator (Kopf) and positioned 0.5 mm above the pial surface. The analog output of the flowmeter was fed into a DC amplifier (Grass) and displayed on the polygraph. To avoid pulsatile variations in the flow signal a long time constant was used (5 s). After a IO- to 20-min stabilization period, probe position and reactivity of the preparation was tested at each site by determining the cerebrovascular reactivity to hypercapnia (12, 17). Once a suitable placement was obtained, the probe was left at that site for the duration of the experiment. At the end of the experiment the heart was stopped by an intravenous bolus injection of saturated KCl, and the zero level for CBF was recorded. Changes in CBF were calculated as percentage of the baseline value. Experimental protocol. After completion of surgical procedures and placement of the LDF probe the rate of administration of halothane was reduced to 1%. This concentration of halothane is virtually identical to its minimal alveolar concentration (32) and is more than adequate for maintenance of anesthesia. Furthermore, rats were not paralyzed so that the level of anesthesia could be monitored by testing cornea1 reflexes and motor responses to tail pinch. An electrode mounted on a micromanipulator with a 10 degree posterior inclination was positioned on the calamus scriptorius and the stereotaxic coordinates recorded as stereotaxic zero (24). The electrode was then moved 5 mm rostra1 and 0.8 mm lateral to zero, lowered into the cerebellum until the vertical stereotaxic zero was reached, and left at that site until the exploration for the FN was begun. After blood gases were adjusted, procedures for localization of the FN were started. As described elsewhere (24), the electrode was withdrawn in 0.5-mm steps, and at each step exploratory stimuli, consisting of 8-s trains of 0.5-ms pulses at 50 Hz and with a current intensity of lo-20 PA, were delivered. An active site in FN was defined as one in which stimulation resulted in a stimulus-locked IO-20 mmHg AP elevation. Once the most active site in FN was localized the electrode was left there. The spinal cord was then transected and the AP maintained between 90 and 110 mmHg by continuous infusion of phenylephrine, as previously described (17, 24) (Table I). After spinal cord transection, stability of the level of anesthesia was assessed by verifying the absence of cornea1 reflexes and spontaneous vibrissal movements. Approximately 30 min after completion of the transection, blood gases were measured again and adjusted. Arterial PCO~ (mmHg), PO, (mmHg), and pH in the rat studied were, respectively, 34.8 t 0.5, 358 zt 23, and 7.474 & 0.01 (mean t SE; n = 25). The PO, of the animals was high. However, this degree of
Table 1. Mean arterial pressure at the time of cerebral blood flow measurement during stimulation of the fastigial nucleus or pontine reticular formation Mean FN stimulation
Ringer Methylene blue Nitro-L-arginine Nitro-D-arginine Values formation.
98t2 lOOt3 lq2+1 98t2
Arterial n
27 22 14 7
Pressure,
mmHg
PRF
stimulation
n
92&l 96k2 95t4
9 9 9
are means f: SE. FN, fastigial nucleus; PRF, pontine reticular P > 0.05 by analysis of variance and Tukey’s test.
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hyperoxia does not affect cerebrovascular tone (31). After AP and blood gases were in a steady state, stimulation of the FN or PRF was started. For stimulation of the FN, stimuli consisted of intermittent (I s on/l s off) trains of negative square waves at 50 Hz and with a current intensity of 50-100 PA. For stimulation of the PRF, the electrode was lowered 1.5-2.0 mm below horizontal zero, and intermittent trains of stimuli were delivered at 100 PA and 100 Hz. Several responses to FN and PRF stimulation were first elicited while the cranial window was superfused with normal Ringer. Once stable and reproducible increases in CBF were obtained, superfusion with the test drugs commenced. The arginine analogues N”-nitro-L-arginine (Sigma) and N”-nitro-D-arginine (D-NA) (Serva) and the soluble guanylyl cyclase inhibitor methylene blue (MB) (Sigma) were topically superfused on the exposed cortex. Solutions were prepared daily. L- and D-NA (1 mM) were dissolved in Ringer acidified by bubbling CO, for 15-20 min. Solutions were applied to the brain after their pH rose to the range of pH of 7.3-7.4. MB was dissolved in Ringer, and the solution was filtered and applied at a concentration of 1 mM. CBF responses to FN and PRF stimulation were then elicited at various time intervals after the start of the superfusion. Maximal effects on the responses evoked from FN stimulation were observed after 45-60 min of superfusion (see below). In experiments in which CBF was measured by the [14C]iodoantipyrine technique with autoradiography, it was found that topical superfusion of L-NA decreases CBF in an area of cortex 3-4 mm wide and 2-3 mm deep (unpublished observations). Therefore, the volume of cortex involved by L-NA is well within the spatial range of monitoring of CBF by the laser-Doppler probe (1 mm3) (see Ref. 17 for references). Data analysis. Data are expressed as means & SE. Comparisons among multiple groups were statistically evaluated by the analysis of variance and the Tukey’s test as a post hoc procedure (Systat). Two-group comparisons were evaluated by the Student’s t test. RESULTS
Effect of MB on the cerebrovasodilation elicited by FN or PRF stimulation. NO produces vascular relaxation by activating soluble guanylyl cyclase (18). Therefore, it was first investigated whether the cerebrovasodilation elicited by FN stimulation is influenced by MB, an inhibitor of cGMP mediated responses (8). During topical application of normal Ringer, stimulation of the FN (100 PA; 50 Hz; 1 s on/l s off) produced the well-established increase in neocortical CBF (+90 t 7%; n = 27) (Fig. 1, Table 2). Topical application of the guanylyl cyclase inhibitor MB (1 mM) did not affect resting CBF (before MB: 10 t 2 perfusion units; after MB: 10 t 2; P > 0.05; paired t test). However, this agent attenuated the vasodilation by 77 t 3% (n = 22; P < 0.001; analysis of variance and Tukey’s test) (Fig. 1; Table 2). The effect of MB was time dependent, in that the magnitude of the attenuation increased as the length of the superfusion increased (Fig. 2). Maximal effects occurred after -45 min of superfusion. This progressive attenuation of the CBF increases was not a consequence of a deterioration of the CBF response to FN stimulation. As shown in Fig. 2, the increase in CBF elicited by FN stimulation, in five separate rats, was stable over a corresponding period of time. In contrast to the FN, MB had no effect on the increases in CBF elicited by stimulation of the PRF: PRF stimulation increased CBF by 127 t 18% (n = 9) during superfusion with Ringer and by 98 t 18% (n = 9) during superfusion with MB (P >
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250 0 Methylene A Ringer
blue
:;, 100
FN stimulation (1 OOpA-50Hz)
2 min
Fig. 1. Effect of topical application of methylene blue (1 mM) or nitroL-arginine (1 mM) on the increases in cerebral blood flow (CBF) elicited by fastigial nucleus (FN) stimulation. Rats were anesthetized and their spinal cord transected. After spinal cord transection arterial pressure was maintained by continuous infusion of phenylephrine. Drugs were superfused over a cranial window drilled through the parietal bone. CBF was monitored at the site of superfusion by a laser-Doppler probe. During Ringer superfusion, FN stimulation increased CBF -100% of control (A). During superfusion with methylene blue (B) or nitroarginine (C) the increases in CBF were substantially attenuated.
Table 2. Effect of methylene blue, nitro-L-arginine or nitro-o-arginine on the increases in cerebral blood flow (75 of control) elicited by electrical stimulation of the fastigial nucleus or the pontine reticular formation
FN stimulation
n PRF
stimulation
n
Ringer
Methylene Blue (1 mM)
Nitro-Larginine (1 mM)
Nitro-Darginine (1 mM)
190&7 27 228t18
121&3* 22 198+18”f
130t3* 14 204t14
184+7t 7
9
Values are means t SE. FN, fastigial formation. * P < 0.001 from Ringer; “f Tukey’s test).
9
9 nucleus;
PRF,
P > 0.05 (analysis
pontine reticular of variance and
0.05) (Fig. 3; Table 2). Effect of arginine analogues on the cerebrovasodilation elicited by FN or PRF stimulation. In these experiments the effect of the inhibitor of NO synthesis L-NA was studied. L-NA (1 mM) produced a significant decrease in resting CBF (-16 t 7; P < 0.02; paired t test). Application of this agent reduced the increases in CBF elicited by FN stimulation by 67 t 3% (n = 14; P < 0.001 from Ringer) (Fig. 1, Table 2). In contrast, application of the inactive stereoisomer D-NA failed to influence the increasesin CBF (+84 t 7%; n = 7; P > 0.05 from Ringer). L-NA did not significantly affect the increases in CBF elicited by PRF stimulation (+104 t 14%; P > 0.05; n = 9). DISCUSSION
This study sought to determine whether NO participates in the cholinergic cerebrovasodilation elicited by FN stimulation. To achieve this goal the inhibitor of NO
0
15 Time
30
45
(min)
Fig. 2. Time course of the effect of methylene blue (MB) on the increase in CBF elicited by FN stimulation. The attenuation of the CBF increase became apparent at I5 min and was maximal at 45 min (open squares) (P < 0.05; analysis of variance and Tukey’s test). During MB superfusion CBF remained stable. The attenuation is not a consequence of deterioration of the preparation as, in separate rats (n = 5), the increases in CBF elicited by FN stimulation were stable in time (solid triangles) (P > 0.05).
2 min PRF stimulation (lOOpA-1OOHz)
Fig. 3. Effect of 1 mM nitro-L-arginine (L-NA; B) vs. control (A) on the increases in CBF elicited by stimulation of the pontine reticular formation (PRF). Note that L-NA did not influence the magnitude of the increases in CBF.
synthesis L-NA and the soluble guanylyl cyclase inhibitor MB were used. It was found that topical superfusion with L-NA or MB substantially attenuated the local increases in CBF while application of D-NA, the inactive stereoisomer of nitroarginine, did not affect the vasodilation. Furthermore, MB or L-NA did not influence the magnitude of the cerebrovasodilation elicited by stimulation of the PRF. The findings indicate that the cerebrovasodilation elicited by FN stimulation depends on NO production and activation of soluble guanylyl cyclase. The attenuation of the cerebrovasodilation by MB and L-NA cannot be a consequence of differences in AP or blood gases among animals as these parameters were carefully controlled and did not differ among experimental groups. Similarly, the effect of MB and L-NA is unlikely to be due to a nonspecific action of these agents resulting in a reduced reactivity of CBF to vasodilating stimuli as L-NA or MB did not influence the increases in CBF elicited by stimulation of the PRF (this study) or papaverine (12). It is also unlikely that the attenuation of the vasodilation is due to an action of these drugs on cerebral metabolism. First, NO-synthase inhibition does not affect resting cerebral glucose utilization (unpublished observations). Second, these drugs do not attenuate the cerebrovasodilation elicited by PRF stimulation, a
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cerebrovascular effect highly sensitive to agents that decrease cerebral metabolism (16). Finally, the attenuation of the CBF response elicited by FN stimulation cannot be an artifact of the technique used to measure CBF. We have previously shown that the cerebrovascular effects of FN stimulation as assessed by LDF are identical to those determined by using more established techniques for measuring CBF (17). In addition, the accuracy of LDF in monitoring CBF changes has been demonstrated in a wide variety of experimental paradigms (see Ref. 17 for references). L-NA or MB was topically superfused over the brain surface for prolonged periods of time. It is conceivable that these agents diffuse into the tissue and subarachnoid space to involve the underlying brain parenchyma as well as more remote sites. Therefore, an important question in this study concerns the site of action of MB and L-NA. Topical application studies in the pial microcirculation have shown that MB can block local endothelium dependent responses within 5-7 min of application (9, 20, 27). We have found that the effect of MB and L-NA on the local increase in CBF elicited by FN stimulation requires longer application periods (30-45 min). This observation indicates that the site of action of MB and L-NA is not restricted to the pial microcirculation at the site of superfusion but may also involve distant sites. Further studies using quantitative autoradiography will be helpful in determining whether the attenuation of the cerebrovasodilation is restricted to the site of superfusion or whether these effects are more widespread. A somewhat related question concerns the cellular source of NO. In brain, NO can be produced in neurons, their processes, astrocytes, and endothelial cells (6, 22). Furthermore, NO can be released from perivascular nerves originating from cranial autonomic ganglia (34). It is unlikely that NO is released from perivascular nerves during FN stimulation because these nerves do not participate in the cerebrovasodilation (26, unpublished observation). One likely source of NO is the endothelium. NO could be produced in endothelial cells of local microvessels in response to ACh or other neurotransmitters released extracellularly or perivascularly. However, for this mechanism to operate ACh must diffuse through the vessel wall and reach the endothelium to stimulate NO production. Another possibility is that NO is released from the endothelium of larger vessels in response to increased flow. In this case it must be postulated that FN stimulation produces an initial local vasodilatory response that is independent of NO. This local vascular response is then propagated retrogradely toward larger arteries resulting in vasodilation of these vessels (30). Increased shear stress in these larger arteries releases NO and produces vasodilatation, which in turn amplifies the vascular response (28). In support of this hypothesis is the observation that after MB or L-NA FN stimulation still elicits an increase in CBF, indicating that a small component of the response is independent of NO. While the possibility of NO release from the endothelium of larger vessels during FN stimulation is attractive, whether these mechanisms participate i n the vasodila-
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tory responses of the cerebral circulation has not been established. On the other hand ., NO could be released by local cortical neural elements. Indeed, the neocortical vasodilation elicited by FN stimulation seems to require the presence of a restricted group of local neurons for its expression (13). Recordings of extracellular K+ in neocortex during FN stimulation indicate that active neurons are localized in the intermediate cortical laminae (14). If NO is released from local neurons, two possibilities are likely. One is that local neurons release ACh, which, in turn, could act on other neurons and/or astrocytes stimulating NO production. Alternatively, NO and ACh could be released from the same neurons and NO could act as the mediator of the cerebrovasodilation. The presence of NO-synthase has been reported in the rat cerebral cortex (3). This enzyme is localized in a discrete subset of cortical interneurons that expresses also neuropeptide Y and somatostatin (36). Irrespective of its cellular origin, NO, being short lived, must be produced near the blood vessels on which it will act (6, 21). Indeed, NO has a biological half-life of only few seconds (21). However, the increases in CBF elicited by FN stimulation have a relatively “slow” time course as they reach their peak 50 to 60 s after the beginning of the stimulation and persist beyond the termination of the stimulus (17). If NO is responsible for the vasodilation then it must be postulated that this agent continues to be generated also after termination of the stimulus. The observation that the increases in CBF elicited by FN stimulation have a “slow” time course (17, 33) has suggested that the response is mediated by interstitial release of diffusible vasoactive factors rather than by activation of direct neurovascular projections, as originally proposed (23, 24, 26). However, the assumption that activation of neurovascular projections produces “fast” changes in CBF has recently been challenged. The increase in cortical CBF elicited by stimulation of the facial nerve, which contains well-described autonomic fibers innervating the cerebral vasculature, has a relatively “slow” time course (7). Therefore, the time course of “neurogenically” mediated changes in CBF may be slower than previously believed. The results of the present study, however, do not elucidate whether direct neurovascular projections are involved in the FN response, because NO could conceivably be released from intrinsic perivascular nerve terminals (34) as well as cortical interneurons (36). There is increasing evidence that NO is involved in regulating vascular tone in the systemic circulation (2 1). Recent studies indicate that NO may participate also in the regulation of the cerebral circulation. While there still is some controversy as to whether in brain vessels NO is the “classical” EDRF (20), this agent seems to be involved in fundamental responses characteristic of the cerebral circulation, such as the cerebrovasodilation elicited by hypercapnia (12). Furthermore, NO appears to mediate the cortical cerebrovasodilation elicited by stimulation of the basal forebrain cholinergic system (2). The present study adds further evidence supporting the notion that NO may be an important molecular messenger also in the cerebral circulation. However, NO is not involved in all
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the vasodilatory responses of the cerebral circulation. For example, it has been shown here that NO does not participate in the elevations in CBF elicited by stimulation of the reticular formation. The increases in CBF associated with sti .mulation of the brain stem reticula .r formation are not cholinergically media .ted (17) and are presumably “secondary” to a global increase in metabolism (16). It is therefore conceivable that NO is not involved in the cerebrovasodilation elicited by arousal and global neuronal activation. Whether NO participates in the cerebrovasodilation elicited by focal neuronal activation, such as that evoked by stimulation of somatosensory or visual pathways (4, 35), remains to be determined. In conclusion, this study demonstrates that the increase in neocortical CBF elicited by stimulation of the FN is mediated, in great part, by arginine-derived NO. Although the cellular source of NO and the segment of the cerebral vasculature on which it acts remain to be determined, the findings support the notion that NO is an important molecular signal with widespread biological actions that may also include the regulation of cerebrovascular tone. This work was supported by grants-in-aid Association (Minnesota) and the Minnesota Portions of this study have been presented Address for reprint requests: C. Iadecola, versity of Minnesota Medical School, Box 295 SE, Minneapolis, MN 55455. Received
26 December
1991; accepted
in final
from the American Heart Medical Foundation. in abstract form (II). Dept. of Neurology, UniUMHC, 420 Delaware St. form
1 May
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and Organ Blood Flow. Regulation of Circulation to Individual Vascular Beds. Bethesda, MD: Am. Physiol. Sot., 1983, sect. 2, vol. III, pt. 1, chapt. 5, p. 137-182. Iadecola, C. EDRF participates in the neocortical vasodilation elicited by stimulation of the fastigial nucleus. Sot. Neurosci. Abstr. 17: 27, 1991. Iadecola, C. Does nitric oxide mediate the cerebrovasodilation elicited by hypercapnia ? Proc. Natl. Acad. Sci. USA 89: 39133916, 1992. Iadecola, C., S. Arneric, H. Baker, L. Tucker, and D. Reis. Role of local neurons in the cerebrocortical vasodilation elicited from cerebellum. Am. J. Physiol. 252 (Regulatory Integrative Comp. Physiol. 21): R1082-R1091, 1987. Iadecola, C., and R. P. Kraig. Elevations in neocortical interstitial K+ elicited from the cerebellar fastigial nucleus in rat. Brain Res. 563: 273-277, 1991. Iadecola, C., S. Mraovitch, M. Meeley, and D. Reis. Lesions of the basal forebrain in rat selectively impair the cortical vasodilation elicited from cerebellar fastigial nucleus. Bruin Res. 279: 41-52, 1983. Iadecola, C., M. Nakai, S. Mraovitch, D. A. Ruggiero, L. W. Tucker, and D. J. Reis. Global increase in cerebral metabolism and blood flow produced by focal electrical stimulation of dorsal medullary reticular formation in rat. Brain Res. 272: lOl114, 1983. Iadecola, C., and D. J. Reis. Continuous monitoring of cerebrocortical blood flow during stimulation of the cerebellar fastigial nucleus: a study by laser-Doppler flowmetry. J. Cereb. Blood Flow Metab. 10: 608-617, 1990. Ignarro, L. J. Heme-dependent activation of guanylate cyclase by nitric oxide: a novel signal transduction mechanism. Blood Vessels 28: 67-73, 1991. Dwyer, M. A., D. S. Bredt, and S. H. Snyder. Nitric oxide synthase: irreversible inhibition by L-NG-nitroarginine in brain in vitro and in vivo. Biochem. Biophys. Res. Commun. 176: 11361141, 1991. Marshall, J. J., E. P. Wei, and H. A. Kontos. Independent blockade of cerebral vasodilation from acetylcholine and nitric oxide. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H847-H854, 1988. Moncada, S., R. M. J. Palmer, and E.‘A. Higgs. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43: 109-142, 1991. Murphy, S., R. L. Minor, Jr., G. Welk, and D. G. Harrison. Evidence for an astrocyte-derived vasorelaxing factor with properties similar to nitric oxide. J. Neurochem. 55: 349-351, 1990. Nakai, M., C. Iadecola, D. Ruggiero, L. Tucker, and D. Reis. Electrical stimulation of cerebellar fastigial nucleus increases cerebrocortical blood flow without change in local metabolism: evidence for an intrinsic system in brain for primary vasodilation. Brain Res. 260: 35-49, 1983. Nakai, M., C. Iadecola, and D. Reis. Global cerebral vasodilation by stimulation of rat fastigial cerebellar nucleus. Am. J. Physiol. 243 ( Heart Circ. Physiol. 12): H226-H235, 1982. Palmer, R., D. Ashton, and S. Moncada. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature Lond. 333: 664-666, 1988. Reis, D., C. Iadecola, E. MacKenzie, M. Mori, M. Nakai, and L. Tucker. Primary and metabolically coupled cerebrovascular dilation elicited by stimulation of two intrinsic systems of brain. In: Cerebral Blood Flow: Effects of Nerves and Neurotransmitters, edited by D. Heistad and M. Marcus. New York: Elsevier/ North Holland, 1982, p. 475-484. Rosenblum, W. I. Endothelial dependent relaxation demonstrated in vivo in cerebral arterioles. Stroke 17: 494-497, 1986. Rubanyi, G. M., J. C. Romero, and P. M. Vanhoutte. Flowinduced release of endothelium-derived relaxing factor. Am. J. Physiol. 250 (Heart Circ. Physiol. 19): Hll45-H1149, 1986. Sakuma, I., D. J. Stuehr, S. S. Gross, C. Nathan, and R. Levi. Identification of arginine as a precursor of endotheliumderived relaxing factor. Proc. Natl. Acad. Sci. USA 85: 8664-8667, 1988. Segal, S. S., and B. R. Duling. Flow control among microvessels coordinated by intercellular conduction. Science Wash. DC 234: 868-870, 1986.
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NEUROGENIC
31. Shinozuka,
T., E. M. Nemoto,
nisms of cerebrovascular ate hypoxia in the rat. 1989.
32. Stimpfel,
and P. M. Winter.
O2 sensitivity from hyperoxia J. Cereb. Blood Flow Metab.
T. M., and E. L. Gershey.
agents for human safety 2099-2104, 1991.
and animal
33. Talman, W. T., D. M. Nitschke H. Ohta. Cerebrovascular effects tion of fastigial 30): H707-H713,
nucleus. 1991.
CEREBROVASODILATION
Am.
Mechato moder9: 187-195,
recovery
Selecting anesthetic surgery. FASEB J. 5:
Dragon,
D. D. Heistad,
AND 34.
35.
and
produced by electrical stimulaJ. Physiol. 261 (Heart Circ. Physiol.
36.
NITRIC
OXIDE
R1161
Toda,
N., and T. Okamura. Mechanism underlying the response to vasodilator nerve stimulation in isolated dog and monkey cerebral arteries. Am. J. Physiol. 259 (Heart Circ. Physiol. 28): H1511-H1517, 1990. Ueki, M., F. Linn, and K.-A. Hossmann. Functional activation of cerebral blood flow and metabolism before and after global ischemia of rat brain. J. Cereb. Blood Flow Metab. 8: 486-494, 1988. Vincent, S. R., and H. Kimura. Histochemical mapping of nitric oxide synthase in the rat brain. Neuroscience 46: 755-784, 1992.
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IADECOLA of Neurology,
University
of Minnesota
Iadecola, Costantino. Nitric oxide participates in the cerebrovasodilation elicited from cerebellar fastigial nucleus. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1156R1161, 1992.-The endothelium-derived relaxing factor, probably NO, is a potent vasodilator that mediates the vasodilating action of acetylcholine (ACh). We studied whether NO participates in the cholinergic cerebrovasodilation elicited by stimulation of the cerebellar fastigial nucleus (FN). Rats were anesthetized with halothane and ventilated. FN or pontine reticular formation (PRF) were stimulated through microelectrodes. Hypertension was prevented by spinal cord transection with arterial pressure maintained by intravenous phenylephrine. Cerebral blood flow (CBF) was continuously monitored through a cranial window over the sensory cortex by a laser-Doppler probe. The window was superfused with Ringer solution (pH 7.3-7.4; 37°C). During Ringer superfusion FN stimulation (100 ,uA; 50 Hz) increased CBF by 90 t 7% (n = 27; P < 0.001, analysis of variance and Tukey’s test) and PRF stimulation (100 PA; 100 Hz) by 128 t 18% (P < 0.001; n = 9). Superfusion with the guanylyl cyclase inhibitor methylene blue (MB) (1 mM) attenuated the CBF increase elicited by FN stimulation by 77 t 3% (n = 22; P < 0.001). MB did not affect the CBF increase elicited by PRF stimulation (+98 t 18%; n = 9; P > 0.05). Similarly, superfusion with the NO-synthase inhibitor nitro-L-arginine (L-NA) attenuated the CBF increase elicited by FN stimulation (-67 t 3%; n = 14; P < 0.001 from Ringer) but not PRF stimulation (P > 0.05; n = 9). The CBF increases elicited by FN stimulation were not affected by the inactive isomer of nitroarginine, D-NA (P > 0.05; n = 7). Thus the neocortical vasodilation elicited by FN stimulation is substantially attenuated by inhibition of NO synthesis or inhibition of the enzyme mediating NO action. The results indicate that the vasodilation elicited by FN stimulation is mediated, for the most part, by arginine-derived NO and suggest that NO may be important also in the regulation of the cerebral circulation. cerebellum; cerebral blood flow; methylene blue; nitroarginine; endothelium-derived relaxing factor STIMULATION of the cerebellar fastigial nucleus (FN) increases cerebral blood flow (CBF) globally through neural pathways entirely contained within the brain (13, 15, 24, 26). The increases in CBF are greatest in regions of the cerebral cortex, wherein the vasodilation is not associated with increased glucose utilization (23). In neocortex, the increase in CBF is substantially attenuated by topical application of atropine (I), a finding indicating that the vasodilation is mediated by local release of acetylcholine (ACh). While it is well established that ACh is a powerful cerebrovascular vasodilator (lo), the local mechanisms by which ACh produces vascular relaxation during FN stimulation remain to be elucidated. There is increasing evidence that the vascular relaxation elicited by ACh is mediated by an endothelium-derived relaxing factor (EDRF) (5, 9, 20, 27), a labile substance that is probably nitric oxide (NO) or a closely related nitroso compound (2 I). It is therefore conceivable that during FN stimulation ACh mediates the cerebrovasodilation through NO. ELECTRICAL
R1156
0363-6119/92
$2.00
Copyright
Medical
School, Minneapolis,
Minnesota
55455
In the present study we sought to determine whether NO participates in the cerebrovasodilation elicited by FN stimulation. NO is synthesized from L-arginine by the enzyme NO-synthase (3, 25, 29), whose activity can be inhibited by N”-substituted arginine analogues such as nitroarginine (L-NA) (19, 21, 29). Furthermore, NO exerts its vasodilating action by activating soluble guanylyl cyclase and increasing 3’,5’-cyclic guanosine monophosphate (cGMP) production in smooth muscles (18). We therefore studied whether inhibition of NO synthesis by NA or inhibition of NO action by the guanylyl cyclase inhibitor methylene blue (MB) attenuates the increase in CBF elicited by FN stimulation. We shall demonstrate that topical superfusion of these agents on the parietal cortex substantially attenuates the local cerebrovasodilation elicited by FN stimulation while the increases in CBF elicited by stimulation of the pontine reticular formation (PRF) are not affected. METHODS Methods for surgical preparation of rats, monitoring of CBF by laser-Doppler flowmetry (LDF), and brain stimulation have been described in detail previously (12-15, 17, 24) and will be summarized below. General procedures. Studies were performed on 25 male Sprague-Dawley rats weighing 290-380 g. Rats were anesthetized with 5% halothane in 100% oxygen, delivered through a facial mask. After induction of anesthesia, halothane was administered at a reduced rate (2%). Catheters were inserted in both femoral arteries and in the right femoral vein, and the trachea was cannulated. Animals were then placed on a stereotaxic frame (Kopf) and artificially ventilated with 100% oxygen by a mechanical ventilator (rodent respirator, Harvard Apparatus). Body temperature was maintained at 37 t 0.5”C using a heating lamp thermostatically controlled by a rectal probe (YSI). One of the arterial catheters was used for continuous recording of arterial pressure (AP), mean AP, and heart rate on a chart recorder (Grass), while the other arterial line was used for blood sampling. The venous catheter was used for intravenous administration of phenylephrine after transection of the spinal cord (see below). Arterial Pco~, PO,, and pH were measured at multiple times on 100 ~1 of blood using a blood gas analyzer (Corning 178). Spinal cord transection. To prevent the effect on CBF of the hypertension elicited by FN or PRF stimulation, the spinal cord was transected at the first cervical segment. As described elsewhere (17, 24), the spinal cord was exposed at the C,-C, junction by removing the posterior lamina of the atlas. After removing the dura, the cord was severed at C1, and a small segment below C, was removed by suction. Just before the procedure, an intravenous infusion of phenylephrine (1.3-6.4 pg/min) was started to counteract the fall in AP that occurs after spinal cord transection. The completeness of the transection was verified at autopsy. Monitoring of CBF by laser-Doppler flowmetry. LDF was performed using a Vasamedic flowmeter (TSI, St. Paul, MN) (12, 14). A 3 x 3 mm cranial window was drilled at a site 2-3 mm lateral and 1-2 mm caudal to bregma. The underlying cortex
0 1992 the American
Physiological
Society
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CEREBROVASODILATION
corresponds to the leg area of the primary sensory cortex (14). The dura was carefully removed and the craniotomy site was continuously superfused at a rate of 0.33 ml/min with Ringer warmed at 37°C and aerated with 95% 0, and 5% CO, (pH 7.3-7.4). The composition of the Ringer (in mM) was 143.5 Na+, 3.0 K+, 1.5 Ca2+, 1.4 Mg2+, 115 Cl-, 26.4 HCO;, 9.6 gluconate, and 5.0 glucose (14). The LDF probe (tip diameter 0.8 mm) was mounted on a micromanipulator (Kopf) and positioned 0.5 mm above the pial surface. The analog output of the flowmeter was fed into a DC amplifier (Grass) and displayed on the polygraph. To avoid pulsatile variations in the flow signal a long time constant was used (5 s). After a IO- to 20-min stabilization period, probe position and reactivity of the preparation was tested at each site by determining the cerebrovascular reactivity to hypercapnia (12, 17). Once a suitable placement was obtained, the probe was left at that site for the duration of the experiment. At the end of the experiment the heart was stopped by an intravenous bolus injection of saturated KCl, and the zero level for CBF was recorded. Changes in CBF were calculated as percentage of the baseline value. Experimental protocol. After completion of surgical procedures and placement of the LDF probe the rate of administration of halothane was reduced to 1%. This concentration of halothane is virtually identical to its minimal alveolar concentration (32) and is more than adequate for maintenance of anesthesia. Furthermore, rats were not paralyzed so that the level of anesthesia could be monitored by testing cornea1 reflexes and motor responses to tail pinch. An electrode mounted on a micromanipulator with a 10 degree posterior inclination was positioned on the calamus scriptorius and the stereotaxic coordinates recorded as stereotaxic zero (24). The electrode was then moved 5 mm rostra1 and 0.8 mm lateral to zero, lowered into the cerebellum until the vertical stereotaxic zero was reached, and left at that site until the exploration for the FN was begun. After blood gases were adjusted, procedures for localization of the FN were started. As described elsewhere (24), the electrode was withdrawn in 0.5-mm steps, and at each step exploratory stimuli, consisting of 8-s trains of 0.5-ms pulses at 50 Hz and with a current intensity of lo-20 PA, were delivered. An active site in FN was defined as one in which stimulation resulted in a stimulus-locked IO-20 mmHg AP elevation. Once the most active site in FN was localized the electrode was left there. The spinal cord was then transected and the AP maintained between 90 and 110 mmHg by continuous infusion of phenylephrine, as previously described (17, 24) (Table I). After spinal cord transection, stability of the level of anesthesia was assessed by verifying the absence of cornea1 reflexes and spontaneous vibrissal movements. Approximately 30 min after completion of the transection, blood gases were measured again and adjusted. Arterial PCO~ (mmHg), PO, (mmHg), and pH in the rat studied were, respectively, 34.8 t 0.5, 358 zt 23, and 7.474 & 0.01 (mean t SE; n = 25). The PO, of the animals was high. However, this degree of
Table 1. Mean arterial pressure at the time of cerebral blood flow measurement during stimulation of the fastigial nucleus or pontine reticular formation Mean FN stimulation
Ringer Methylene blue Nitro-L-arginine Nitro-D-arginine Values formation.
98t2 lOOt3 lq2+1 98t2
Arterial n
27 22 14 7
Pressure,
mmHg
PRF
stimulation
n
92&l 96k2 95t4
9 9 9
are means f: SE. FN, fastigial nucleus; PRF, pontine reticular P > 0.05 by analysis of variance and Tukey’s test.
AND
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OXIDE
Rl157
hyperoxia does not affect cerebrovascular tone (31). After AP and blood gases were in a steady state, stimulation of the FN or PRF was started. For stimulation of the FN, stimuli consisted of intermittent (I s on/l s off) trains of negative square waves at 50 Hz and with a current intensity of 50-100 PA. For stimulation of the PRF, the electrode was lowered 1.5-2.0 mm below horizontal zero, and intermittent trains of stimuli were delivered at 100 PA and 100 Hz. Several responses to FN and PRF stimulation were first elicited while the cranial window was superfused with normal Ringer. Once stable and reproducible increases in CBF were obtained, superfusion with the test drugs commenced. The arginine analogues N”-nitro-L-arginine (Sigma) and N”-nitro-D-arginine (D-NA) (Serva) and the soluble guanylyl cyclase inhibitor methylene blue (MB) (Sigma) were topically superfused on the exposed cortex. Solutions were prepared daily. L- and D-NA (1 mM) were dissolved in Ringer acidified by bubbling CO, for 15-20 min. Solutions were applied to the brain after their pH rose to the range of pH of 7.3-7.4. MB was dissolved in Ringer, and the solution was filtered and applied at a concentration of 1 mM. CBF responses to FN and PRF stimulation were then elicited at various time intervals after the start of the superfusion. Maximal effects on the responses evoked from FN stimulation were observed after 45-60 min of superfusion (see below). In experiments in which CBF was measured by the [14C]iodoantipyrine technique with autoradiography, it was found that topical superfusion of L-NA decreases CBF in an area of cortex 3-4 mm wide and 2-3 mm deep (unpublished observations). Therefore, the volume of cortex involved by L-NA is well within the spatial range of monitoring of CBF by the laser-Doppler probe (1 mm3) (see Ref. 17 for references). Data analysis. Data are expressed as means & SE. Comparisons among multiple groups were statistically evaluated by the analysis of variance and the Tukey’s test as a post hoc procedure (Systat). Two-group comparisons were evaluated by the Student’s t test. RESULTS
Effect of MB on the cerebrovasodilation elicited by FN or PRF stimulation. NO produces vascular relaxation by activating soluble guanylyl cyclase (18). Therefore, it was first investigated whether the cerebrovasodilation elicited by FN stimulation is influenced by MB, an inhibitor of cGMP mediated responses (8). During topical application of normal Ringer, stimulation of the FN (100 PA; 50 Hz; 1 s on/l s off) produced the well-established increase in neocortical CBF (+90 t 7%; n = 27) (Fig. 1, Table 2). Topical application of the guanylyl cyclase inhibitor MB (1 mM) did not affect resting CBF (before MB: 10 t 2 perfusion units; after MB: 10 t 2; P > 0.05; paired t test). However, this agent attenuated the vasodilation by 77 t 3% (n = 22; P < 0.001; analysis of variance and Tukey’s test) (Fig. 1; Table 2). The effect of MB was time dependent, in that the magnitude of the attenuation increased as the length of the superfusion increased (Fig. 2). Maximal effects occurred after -45 min of superfusion. This progressive attenuation of the CBF increases was not a consequence of a deterioration of the CBF response to FN stimulation. As shown in Fig. 2, the increase in CBF elicited by FN stimulation, in five separate rats, was stable over a corresponding period of time. In contrast to the FN, MB had no effect on the increases in CBF elicited by stimulation of the PRF: PRF stimulation increased CBF by 127 t 18% (n = 9) during superfusion with Ringer and by 98 t 18% (n = 9) during superfusion with MB (P >
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R1158
NEUROGENIC
CEREBROVASODILATION
AND
NITRIC
OXIDE
250 0 Methylene A Ringer
blue
:;, 100
FN stimulation (1 OOpA-50Hz)
2 min
Fig. 1. Effect of topical application of methylene blue (1 mM) or nitroL-arginine (1 mM) on the increases in cerebral blood flow (CBF) elicited by fastigial nucleus (FN) stimulation. Rats were anesthetized and their spinal cord transected. After spinal cord transection arterial pressure was maintained by continuous infusion of phenylephrine. Drugs were superfused over a cranial window drilled through the parietal bone. CBF was monitored at the site of superfusion by a laser-Doppler probe. During Ringer superfusion, FN stimulation increased CBF -100% of control (A). During superfusion with methylene blue (B) or nitroarginine (C) the increases in CBF were substantially attenuated.
Table 2. Effect of methylene blue, nitro-L-arginine or nitro-o-arginine on the increases in cerebral blood flow (75 of control) elicited by electrical stimulation of the fastigial nucleus or the pontine reticular formation
FN stimulation
n PRF
stimulation
n
Ringer
Methylene Blue (1 mM)
Nitro-Larginine (1 mM)
Nitro-Darginine (1 mM)
190&7 27 228t18
121&3* 22 198+18”f
130t3* 14 204t14
184+7t 7
9
Values are means t SE. FN, fastigial formation. * P < 0.001 from Ringer; “f Tukey’s test).
9
9 nucleus;
PRF,
P > 0.05 (analysis
pontine reticular of variance and
0.05) (Fig. 3; Table 2). Effect of arginine analogues on the cerebrovasodilation elicited by FN or PRF stimulation. In these experiments the effect of the inhibitor of NO synthesis L-NA was studied. L-NA (1 mM) produced a significant decrease in resting CBF (-16 t 7; P < 0.02; paired t test). Application of this agent reduced the increases in CBF elicited by FN stimulation by 67 t 3% (n = 14; P < 0.001 from Ringer) (Fig. 1, Table 2). In contrast, application of the inactive stereoisomer D-NA failed to influence the increasesin CBF (+84 t 7%; n = 7; P > 0.05 from Ringer). L-NA did not significantly affect the increases in CBF elicited by PRF stimulation (+104 t 14%; P > 0.05; n = 9). DISCUSSION
This study sought to determine whether NO participates in the cholinergic cerebrovasodilation elicited by FN stimulation. To achieve this goal the inhibitor of NO
0
15 Time
30
45
(min)
Fig. 2. Time course of the effect of methylene blue (MB) on the increase in CBF elicited by FN stimulation. The attenuation of the CBF increase became apparent at I5 min and was maximal at 45 min (open squares) (P < 0.05; analysis of variance and Tukey’s test). During MB superfusion CBF remained stable. The attenuation is not a consequence of deterioration of the preparation as, in separate rats (n = 5), the increases in CBF elicited by FN stimulation were stable in time (solid triangles) (P > 0.05).
2 min PRF stimulation (lOOpA-1OOHz)
Fig. 3. Effect of 1 mM nitro-L-arginine (L-NA; B) vs. control (A) on the increases in CBF elicited by stimulation of the pontine reticular formation (PRF). Note that L-NA did not influence the magnitude of the increases in CBF.
synthesis L-NA and the soluble guanylyl cyclase inhibitor MB were used. It was found that topical superfusion with L-NA or MB substantially attenuated the local increases in CBF while application of D-NA, the inactive stereoisomer of nitroarginine, did not affect the vasodilation. Furthermore, MB or L-NA did not influence the magnitude of the cerebrovasodilation elicited by stimulation of the PRF. The findings indicate that the cerebrovasodilation elicited by FN stimulation depends on NO production and activation of soluble guanylyl cyclase. The attenuation of the cerebrovasodilation by MB and L-NA cannot be a consequence of differences in AP or blood gases among animals as these parameters were carefully controlled and did not differ among experimental groups. Similarly, the effect of MB and L-NA is unlikely to be due to a nonspecific action of these agents resulting in a reduced reactivity of CBF to vasodilating stimuli as L-NA or MB did not influence the increases in CBF elicited by stimulation of the PRF (this study) or papaverine (12). It is also unlikely that the attenuation of the vasodilation is due to an action of these drugs on cerebral metabolism. First, NO-synthase inhibition does not affect resting cerebral glucose utilization (unpublished observations). Second, these drugs do not attenuate the cerebrovasodilation elicited by PRF stimulation, a
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NEUROGENIC
CEREBROVASODILATION
cerebrovascular effect highly sensitive to agents that decrease cerebral metabolism (16). Finally, the attenuation of the CBF response elicited by FN stimulation cannot be an artifact of the technique used to measure CBF. We have previously shown that the cerebrovascular effects of FN stimulation as assessed by LDF are identical to those determined by using more established techniques for measuring CBF (17). In addition, the accuracy of LDF in monitoring CBF changes has been demonstrated in a wide variety of experimental paradigms (see Ref. 17 for references). L-NA or MB was topically superfused over the brain surface for prolonged periods of time. It is conceivable that these agents diffuse into the tissue and subarachnoid space to involve the underlying brain parenchyma as well as more remote sites. Therefore, an important question in this study concerns the site of action of MB and L-NA. Topical application studies in the pial microcirculation have shown that MB can block local endothelium dependent responses within 5-7 min of application (9, 20, 27). We have found that the effect of MB and L-NA on the local increase in CBF elicited by FN stimulation requires longer application periods (30-45 min). This observation indicates that the site of action of MB and L-NA is not restricted to the pial microcirculation at the site of superfusion but may also involve distant sites. Further studies using quantitative autoradiography will be helpful in determining whether the attenuation of the cerebrovasodilation is restricted to the site of superfusion or whether these effects are more widespread. A somewhat related question concerns the cellular source of NO. In brain, NO can be produced in neurons, their processes, astrocytes, and endothelial cells (6, 22). Furthermore, NO can be released from perivascular nerves originating from cranial autonomic ganglia (34). It is unlikely that NO is released from perivascular nerves during FN stimulation because these nerves do not participate in the cerebrovasodilation (26, unpublished observation). One likely source of NO is the endothelium. NO could be produced in endothelial cells of local microvessels in response to ACh or other neurotransmitters released extracellularly or perivascularly. However, for this mechanism to operate ACh must diffuse through the vessel wall and reach the endothelium to stimulate NO production. Another possibility is that NO is released from the endothelium of larger vessels in response to increased flow. In this case it must be postulated that FN stimulation produces an initial local vasodilatory response that is independent of NO. This local vascular response is then propagated retrogradely toward larger arteries resulting in vasodilation of these vessels (30). Increased shear stress in these larger arteries releases NO and produces vasodilatation, which in turn amplifies the vascular response (28). In support of this hypothesis is the observation that after MB or L-NA FN stimulation still elicits an increase in CBF, indicating that a small component of the response is independent of NO. While the possibility of NO release from the endothelium of larger vessels during FN stimulation is attractive, whether these mechanisms participate i n the vasodila-
AND
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OXIDE
Rll59
tory responses of the cerebral circulation has not been established. On the other hand ., NO could be released by local cortical neural elements. Indeed, the neocortical vasodilation elicited by FN stimulation seems to require the presence of a restricted group of local neurons for its expression (13). Recordings of extracellular K+ in neocortex during FN stimulation indicate that active neurons are localized in the intermediate cortical laminae (14). If NO is released from local neurons, two possibilities are likely. One is that local neurons release ACh, which, in turn, could act on other neurons and/or astrocytes stimulating NO production. Alternatively, NO and ACh could be released from the same neurons and NO could act as the mediator of the cerebrovasodilation. The presence of NO-synthase has been reported in the rat cerebral cortex (3). This enzyme is localized in a discrete subset of cortical interneurons that expresses also neuropeptide Y and somatostatin (36). Irrespective of its cellular origin, NO, being short lived, must be produced near the blood vessels on which it will act (6, 21). Indeed, NO has a biological half-life of only few seconds (21). However, the increases in CBF elicited by FN stimulation have a relatively “slow” time course as they reach their peak 50 to 60 s after the beginning of the stimulation and persist beyond the termination of the stimulus (17). If NO is responsible for the vasodilation then it must be postulated that this agent continues to be generated also after termination of the stimulus. The observation that the increases in CBF elicited by FN stimulation have a “slow” time course (17, 33) has suggested that the response is mediated by interstitial release of diffusible vasoactive factors rather than by activation of direct neurovascular projections, as originally proposed (23, 24, 26). However, the assumption that activation of neurovascular projections produces “fast” changes in CBF has recently been challenged. The increase in cortical CBF elicited by stimulation of the facial nerve, which contains well-described autonomic fibers innervating the cerebral vasculature, has a relatively “slow” time course (7). Therefore, the time course of “neurogenically” mediated changes in CBF may be slower than previously believed. The results of the present study, however, do not elucidate whether direct neurovascular projections are involved in the FN response, because NO could conceivably be released from intrinsic perivascular nerve terminals (34) as well as cortical interneurons (36). There is increasing evidence that NO is involved in regulating vascular tone in the systemic circulation (2 1). Recent studies indicate that NO may participate also in the regulation of the cerebral circulation. While there still is some controversy as to whether in brain vessels NO is the “classical” EDRF (20), this agent seems to be involved in fundamental responses characteristic of the cerebral circulation, such as the cerebrovasodilation elicited by hypercapnia (12). Furthermore, NO appears to mediate the cortical cerebrovasodilation elicited by stimulation of the basal forebrain cholinergic system (2). The present study adds further evidence supporting the notion that NO may be an important molecular messenger also in the cerebral circulation. However, NO is not involved in all
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R1160
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the vasodilatory responses of the cerebral circulation. For example, it has been shown here that NO does not participate in the elevations in CBF elicited by stimulation of the reticular formation. The increases in CBF associated with sti .mulation of the brain stem reticula .r formation are not cholinergically media .ted (17) and are presumably “secondary” to a global increase in metabolism (16). It is therefore conceivable that NO is not involved in the cerebrovasodilation elicited by arousal and global neuronal activation. Whether NO participates in the cerebrovasodilation elicited by focal neuronal activation, such as that evoked by stimulation of somatosensory or visual pathways (4, 35), remains to be determined. In conclusion, this study demonstrates that the increase in neocortical CBF elicited by stimulation of the FN is mediated, in great part, by arginine-derived NO. Although the cellular source of NO and the segment of the cerebral vasculature on which it acts remain to be determined, the findings support the notion that NO is an important molecular signal with widespread biological actions that may also include the regulation of cerebrovascular tone. This work was supported by grants-in-aid Association (Minnesota) and the Minnesota Portions of this study have been presented Address for reprint requests: C. Iadecola, versity of Minnesota Medical School, Box 295 SE, Minneapolis, MN 55455. Received
26 December
1991; accepted
in final
from the American Heart Medical Foundation. in abstract form (II). Dept. of Neurology, UniUMHC, 420 Delaware St. form
1 May
AND
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