Gacyclidine (Beaufour-Ipsen).

Beaufour-Ipsen is developing gacyclidine (GK-11) for the potential treatment of traumatic brain injury. Phase II clinical trials of the compound for this indication had been completed as of October 1999 and the company is looking for a partnership before undertaking further clinical development for this and, possibly, other indications [344879], [346265], [386763]. Phase II trials for acute spinal cord injury gave disappointing results and development for this indication has been discontinued [344879].

Delayed multidose treatment with nicotinamide extends the degree and duration of neuroprotection by reducing infarction and improving behavioral scores up to two weeks following transient focal cerebral ischemia in Wistar rats.

A single, delayed dose of nicotinamide (NAm) was shown to be protective against focal cerebral ischemia in rats, but the protection was limited to three to seven days following stroke. The investigation reported here was conducted to examine if the use of multiple doses of NAm, administered after the onset of focal cerebral ischemia, would extend the duration of neuroprotection compared with a single dose treatment regimen. Male Wistar rats were subjected to transient focal cerebral ischemia by occluding the right middle cerebral artery (MCAo) for two hours. Following MCAo, motor and sensory behavioral tests were performed daily and the cerebral infarct volumes were measured at two weeks after sacrifice. Each animal was placed into one of four groups that received either normal saline alone (Group S), one (Group A), two (Group B), or three (Group C) doses of NAm (500 mg/kg). Each animal, therefore, received three treatments over two weeks, with the first dose administered intravenously two hours after the onset of MCAo. Single and multiple doses of NAm reduced the infarction (p < 0.01) and improved (p < 0.05) the neurologic sensory and motor behavior when compared with the saline-treated animals up to two weeks after stroke. Moreover, animals that received multiple doses of NAm recuperated full motor function not different from normal, preoperative motor behavior. Delayed treatment with NAm given as multiple doses, therefore, further enhances the extent and duration of neuroprotection by significantly reducing cerebral infarct volumes, improving neurologic behavioral scores, and confers a complete motor recovery up to two weeks from the onset of focal cerebral ischemia in Wistar rats.

Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death.

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programmed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pHi). Exposure to the NO donors SIN-1 and NOC-9 (300 micromol/L) alone rapidly increased genomic DNA fragmentation from 20 +/- 4% to 71 +/- 5% and membrane PS exposure from 14 +/- 3% to 76 +/- 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pHi, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.

Delayed treatment with nicotinamide (Vitamin B(3)) improves neurological outcome and reduces infarct volume after transient focal cerebral ischemia in Wistar rats.

BACKGROUND AND PURPOSE: We have previously shown that nicotinamide (NAm) acutely reduces brain infarction induced by permanent middle cerebral artery occlusion (MCAo) in rats. In this study, we investigate whether NAm may protect against ischemia/reperfusion injury by improving sensory and motor behavior as well as brain infarction volumes in a model of transient focal cerebral ischemia. METHODS: Forty-eight male Wistar rats were used, and transient focal cerebral ischemia was induced by MCAo for 2 hours, followed by reperfusion for either 3 or 7 days. Animals were treated with either intraperitoneal saline or NAm (500 mg/kg) 2 hours after the onset of MCAo (ie, on reperfusion). Sensory and motor behavior scores and body weight were obtained daily, and brain infarction volumes were measured on euthanasia. RESULTS: Relative to treatment with saline, treatment with NAm (500 mg/kg IP) 2 hours after the onset of transient focal cerebral ischemia in Wistar rats significantly improved sensory (38%, P<0.005) and motor (42%, P<0.05) neurological behavior and weight gain (7%, P<0.05) up to 7 days after MCAo. The cerebral infarct volumes were also reduced 46% (P<0.05) at 3 days and 35% (P=0.09) at 7 days after MCAo. CONCLUSIONS: NAm is a robust neuroprotective agent against ischemia/reperfusion-induced brain injury in rats, even when administered up to 2 hours after the onset of stroke. Delayed NAm treatment improved both anatomic and functional indices of brain damage. Further studies are needed to clarify whether multiple doses of NAm will improve the extent and duration of this neuroprotective effect and to determine the mechanism(s) of action underlying the neuroprotection observed. Because NAm is already used clinically in large doses and has few side effects, these results are encouraging for the further examination of the possible use of NAm as a therapeutic neuroprotective agent in the clinical treatment of acute ischemic stroke.

Post-treatment with nicotinamide (vitamin B(3)) reduces the infarct volume following permanent focal cerebral ischemia in female Sprague-Dawley and Wistar rats.

Delayed treatment with nicotinamide (NAm) protects male rats against cerebral ischemia. Since the preponderant use of male animals in stroke research may produce results not applicable to female stroke patients due to gender-related differences, we examined whether delayed NAm treatment could protect female rats against focal cerebral ischemia using a model of permanent middle cerebral artery occlusion (MCAo). NAm (500 mg/kg) given intravenously, 2 h after MCAo, significantly reduced the infarct volume of female Sprague-Dawley (55%, P<0.05) and Wistar rats (60%, P<0.05) rats when compared with saline-injected controls. These studies confirm that NAm is neuroprotective specifically at the dose of 500 mg/kg in rats. The novel findings are that this neuroprotection occurs in female, as well as male rats, and that the neuroprotection observed is more robust when administered as an intravenous bolus compared with intraperitoneal administration.

Mexiletine and magnesium independently, but not combined, protect against permanent focal cerebral ischemia in Wistar rats.

The neuroprotective effect of mexiletine (Mex), a potent Na(+) channel blocker which decreases neuronal energy demands and prevents energy depletion during ischemia, was evaluated in Wistar rats subjected to permanent middle cerebral artery (MCA) occlusion. Postmortem infarct volumes were determined by quantitative image analysis of triphenyltetrazolium (TTC)-stained brain sections. Pretreatment with Mex resulted in a significant infarct volume reduction when administered intraperitoneally, either at the dosage of 50 or 60 mg/kg, 1 hr before MCA occlusion (P < 0.05). Delayed treatment with Mex (50 mg/kg) also had neuroprotective effects when given at 0.5 hr (< 0.05), but not 2-4 hr, after MCA occlusion. Intraarterial administration of MgSO(4) (90 mg/kg), in combination with Mex at 60 mg/kg, showed no additive neuroprotective effect, although each agent independently reduced the MCA occlusion-induced infarction volume (P < 0.05). Our results indicate that a single, acute administration of Mex is neuroprotective against permanent focal cerebral ischemia, but perhaps chronic administration is needed to establish a more effective therapeutic window beyond 0.5 hr. Moreover, the present in vivo data do not favor a combined use of Mg(2+) with Mex for limiting ischemic injury in the brain, since these agents caused cardiopulmonary suppression, which may have led to the loss of the neuroprotective effect of each agent independently.

An in vitro rabbit retina model to study electrophysiologic and metabolic function during and following ischemia.

Most in vitro studies involving neuronal ischemia use biochemical measures and/or cell counting to assess cellular death. We describe an in vitro rabbit retina model in which we measured glucose utilization, lactate production, and light-evoked compound action potentials (CAPs) to assess metabolic and functional recovery following ischemia. Under control conditions, retinal glucose utilization and lactate production (n = 7), as well as CAPs (n = 8) remained quite constant for 6-8 h. During ischemia (glucose reduced from 6 to 1 mM and oxygen from 95 to 15%), glucose utilization and lactate production fell to 50%. CAPs fell to 50% in 3-4 min, and to 0% in 8-10 min. Recovery during 3-4 h of 'return-to-control' was dependent upon the length of ischemia. Glucose utilization recovered to 63% after 1 h (n = 4) and to 18% after 2 h of ischemia (n = 6, P < 0.001). Lactate production recovered to 77% after 1 h (n = 4) and to 54% after 2 h of ischemia (n = 6, P < 0.001). CAPs returned to 51, 15, and 0.13% of the control responses after 0.5 h (n = 7), 1 h (n = 8), and 2 h (n = 5) of ischemia, respectively (P < 0.001). This avascular, blood-brain barrier-free preparation provides an opportunity to use both metabolic and functional criteria to test protection against neuronal ischemia.

Nicotinamide reduces infarction up to two hours after the onset of permanent focal cerebral ischemia in Wistar rats.

Ischemia depletes ATP and initiates cascades leading to irreversible tissue injury. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD+) which increases neuronal ATP concentration and protects against malonate-induced neurotoxicity, trauma and nitric oxide toxicity. We therefore examined whether nicotinamide could protect against stroke, using a model of permanent middle cerebral artery occlusion (MCA) occlusion in Wistar rats. Nicotinamide reduced neuronal infarction in a dose-specific manner. Furthermore, nicotinamide (500 mg/kg) reduced infarcts when administered up to 2 h after the onset of permanent MCA occlusion. The mechanism of action underlying the neuroprotection observed with nicotinamide remains to be clarified. These results are potentially important since nicotinamide is already used clinically, though not in the treatment of stroke.

Neuroprotection against ischemia by metabolic inhibition revisited. A comparison of hypothermia, a pharmacologic cocktail and magnesium plus mexiletine.

Previous studies have suggested that metabolic inhibition is neuroprotective, but little evidence has been provided to support this proposal. Using the in vitro rabbit retina preparation as an established model of the central nervous system (CNS), we measured the rate of glucose utilization and lactate production, and the light-evoked compound action potentials (CAPs) as indices of neuronal energy metabolism and electrophysiologic function, respectively. We examined the effect of three (3) treatments options: hypothermia (i.e., 33 degrees C and 30 degrees C), a six-member pharmacologic "cocktail" (tetrodotoxin (0.1 microM), 2-amino-4-phosphonobutyric acid (20 microM), 2-amino-5-phosphonovaleric acid (1 mM), amiloride (1 mM), magnesium (10 mM) and lithium (10 mM) and the combination of magnesium (Mg2+ 1 mM) and mexiletine (Mex, 300 microM) on in vitro rabbit retinas, to see if there is a correlation between neuronal energy metabolism during ischemia (simulated by the reduction of oxygen from 95% to 15% and glucose from 6 mM to 1 mM), and the subsequent recovery of function. Hypothermia and the "cocktail" significantly inhibited both the rate of glucose utilization and lactate production, whereas Mg2+ and/or Mex showed only a nonsignificant tendency toward a reduction, compared to control retinas. Recovery of light-evoked CAPs was significantly improved in hypothermia- and cocktail-treated retinas, as well as with retinas exposed to the combination of Mg2+ plus Mex, but not with Mg2+ or Mex alone, relative to control retinas. A linear regression analysis of the % recovery of function versus the % reduction in the rate of glucose utilization during ischemia showed a significant correlation (r2 = 0.80, correlation coefficient = 0.9, p < 0.05) between these two parameters. This and other data discussed provide convincing evidence that there is a correlation between metabolic inhibition, achieved during ischemia, and neuroprotection.

Acetylsalicylate administered during simulated ischemia reduces the recovery of neuronal function in the in vitro rabbit retina.

Aspirin is widely used as an analgesic, in the secondary prevention of stroke, and has recently been suggested to be a putative neuroprotective agent, yet whether it acts directly on the central nervous system (CNS) is not yet clarified. We therefore examined the effect of lysine acetylsalicylate (L-ASA, 4-2000 microM) on neuronal function under normal conditions and following 1 h of ischemia using the in vitro rabbit retina preparation. L-ASA inhibited the light-evoked compound action potentials, but not the electroretinogram, in a concentration-dependent manner. In addition, L-ASA (2000 microM, but not 4, 40 or 200 microM) administered during ischemia, reduced the recovery of neuronal function compared to control (untreated) retinas. L-ASA therefore inhibits CNS neurotransmission, but not phototransduction, in a concentration-dependent manner. In addition, high concentration L-ASA impairs the recovery of neuronal function following an ischemic episode.

Induced hypertension improves regional blood flow and protects against infarction during focal ischemia: time course of changes in blood flow measured by laser Doppler imaging.

OBJECTIVE: To characterize changes in regional blood flow (rCBF) during and after a period of arterial occlusion and determine the effect on rCBF and on the extent of infarction when the mean arterial blood pressure is increased during the period of occlusion. METHODS: rCBF in the middle cerebral artery (MCA) territory of rabbits was monitored using laser Doppler perfusion imaging before, during, and after a 1- or 2-hour period of MCA occlusion, and the size of the infarction was assessed by 2,3,5-triphenyltetrazolamine chloride staining after 2 hours of reperfusion. Test animals, the mean arterial blood pressure of which was increased by 65 mm Hg with intravenous phenylephrine during the ischemia, were compared with control animals that remained normotensive. The laser Doppler perfusion imager (Lisca Developments Co., Linköping, Sweden) scanned a 3-cm2 area of cortex with a resolution of 4 mm2 every 15 minutes. RESULTS: MCA occlusion reduced rCBF to 71 +/- 2% of the control level (n = 24, P < 0.001). Hypertension (HTN) restored rCBF to 84 +/- 3% of the control level (n = 12, P < 0.01), but the HTN-induced improvement diminished with time, so that after 1 hour, there was no longer a significant difference between hypertensive and normotensive animals. HTN during the MCA occlusion caused a 97% reduction in infarct size (P < 0.05) in the animals subjected to 1 hour of occlusion but caused only a 45% reduction (P approximately 0.1) in the animals subjected to 2 hours of occlusion. CONCLUSION: This study supports the use of HTN to minimize ischemic injury from short intervals of major intracranial vessel occlusion but fails to demonstrate protection when HTN is maintained during occlusions of more than 1 hour.

Magnesium plus mexiletine inhibit energy usage and protect retinas against ischemia.

We determined whether exogenous Mg2+ and/or mexiletine (Mex), which are reported to be neuroprotective agents, reduced neuronal energy requirements and protected against ischemia, using isolated rabbit retinas. Under non-ischemic conditions, Mex (300 microM) and the combination of Mg2+ (1 mM) plus Mex (300 microM) significantly reduced glucose utilization, by 19% and 31%, respectively. The combination of Mg2+ plus Mex, but not either agent alone, significantly reduced lactate production (by 18%; p < 0.05). When added during 2 h of ischemia (simulated by the reduction of oxygen from 95% to 15% and of glucose from 6 mM to 1 mM), Mg2+ plus Mex improved the recovery of glucose utilization (p < 0.01), lactate production (p < 0.05) and neuronal function (p < 0.05) for 3 h following return to control (post-ischemia/recovery) conditions. Thus reducing energy demands by blocking functions during temporary ischemia, protects neurons from irreversible functional damage.

Avoiding stroke during cerebral arterial occlusion by temporarily blocking neuronal functions in the rabbit.

The temporary occlusion of cerebral vessels is being used with increased frequency in the surgical management of cerebral vascular disease, and this procedure places brain tissue at risk of infarction. Using a modified version of a well-established model of focal cerebral ischemia in the rabbit, we tested the protective effect of a combination of six agents; each agent was selected to temporarily block one or more neuronal functions, hence reducing their metabolic demands. The combination of six agents had been previously shown to protect neurological function against ischemia. Ten male adult New Zealand White rabbits were anesthetized with halothane, and physiological parameters were maintained within normal ranges. A branch of the left external carotid artery was catheterized and the vasculature supplying the left middle cerebral artery (MCA) territory was isolated. Mannitol was infused via the external carotid artery into the left internal carotid artery to open the blood-brain barrier in the territory of the MCA. This infusion was followed by either Ames' medium alone (control) or Ames' medium containing the combination of agents: tetrodotoxin (0.1 micromol/L), 2-amino-4-phosphonobutyric acid (20 mumol/L), 2-amino-5-phosphonovaleric acid (1 mmol/L), amiloride (1 mmol/L), magnesium (10 mmol/L), and lithium (10 mmol/L). Ischemia in the left MCA territory was then induced for 2 hours, followed by 4 hours of reperfusion. Animals pretreated with the combination of agents sustained infarctions that were markedly smaller (mean+/-SEM, 46+/-19.7 mm(3), n=5) than control animals (300+/-46.5 mm(3), n=5, P<.001). We conclude that the strategy of locally delivering a combination of agents designed to temporarily reduce neuronal metabolic demands by temporarily blocking several nonvital neuronal functions, can reduce the infarction induced by a focal reduction in cerebral blood flow in the rabbit.

Nitric oxide produced during ischemia improves functional recovery in the rabbit retina.

We examined the effect of modulating endogenous nitric oxide (NO) production on the recovery of neuronal function from temporary ischemia using a preparation in which blood flow is not a factor. Inhibition of nitric oxide synthase (NOS) during ischemia with L-NA (100 mumol-1) resulted in worse functional recovery compared to D-NA (100 mumol-1)-treated in control retinas (p < 0.01). In contrast, addition of L-Arg (1000 mumol-1) during ischemia, resulted in a concentration-dependent functional improvement (p < 0.05). These results show that inhibition of constitutive NOS is detrimental, whilst the enhancement of endogenous NO production improves the recovery of neuronal function during a period of temporary ischemia in the isolated retina, (an in vitro avascular model of the CNS). Thus, independent of its effects on the vasculature, NO production during temporary ischemia protects neurons from irreversible function damage.

Protection against CNS ischemia by temporary interruption of function-related processes of neurons.

Previous studies have shown that most of the energy consumption of CNS tissue is used for processes that subserve signaling functions of the cells. Since these function-related processes are probably not essential to cell viability, blocking them reversibly with a combination of pharmacologic agents should protect cells from a reduction in energy metabolism. Preliminary experiments to test this hypothesis were performed on isolated rabbit retinas. They were maintained in a newly devised chamber that permitted continuous monitoring of electrophysiological function for > or = 8 h. Ischemia was simulated by a 6-fold reduction in both O2 and glucose. This caused a rapid (t1/2 75 s) and complete loss of the light-evoked response in the optic nerve. Untreated retinas showed full recovery after 1/2 h of deprivation, but only 50% recovery after 1 h and little or no recovery after 2 or 3 h. Retinas exposed during 3 h of deprivation to a combination of six agents that abolished electrophysiologic function and reduced glucose utilization [tetrodotoxin (TTX), 2-amino-4-phosphonobutyric acid (APB), 2-amino-5-phosphonovaleric acid (APV), amiloride, Mg2+, and Li+] showed full recovery. We conclude that reducing energy requirements by blocking functional processes can prevent ischemic damage.

Patterns of peptide-containing perivascular nerves in the circle of Willis: their absence in intracranial arteriovenous malformations.

Using standard immunohistochemical techniques and an improved procedure for whole-mount vascular preparations, the authors describe the pattern and density of innervation of calcitonin gene-related peptide (CGRP)-like, neuropeptide Y (NPY)-like and vasoactive intestinal polypeptide (VIP)-like immunoreactivity in major arteries of postmortem adult human circles of Willis. Calcitonin gene-related peptide-, NPY-, and VIP-LI exhibited a variety of varicose and nonvaricose single axons, and small and large perivascular nerve bundles. Although the density of innervation within each vascular segment was highly variable, the pattern of innervation for each neuropeptide observed was consistent throughout the circle of Willis. With the use of human and rat circles of Willis as positive control preparations, the lack of CGRP-LI, NPY-LI, and VIP-LI in vessel segments taken from five cases of intracranial arteriovenous malformations (AVMs) is also reported. It is concluded that adult human circles of Willis exhibit CGRP-LI, NPY-LI, and VIP-LI perivascular nerves. In addition, intracranial AVMs do not possess these peptide-containing nerves that, in animals, normally mediate neurogenic control in the cerebrovasculature. It is hypothesized that this lack of innervation, and hence neurotrophic influence, may contribute to the development of AVMs.

Nitric oxide modulates light-evoked compound action potentials in the intact rabbit retina.

This study examines the effects of alterations in nitric oxide (NO) production on rabbit retinal function. NG-nitro-L-arginine (L-NA) and L-arginine (L-Arg) inhibited the light-evoked compound action potentials (CAPs) from the optic nerve, but not the electroretinogram (ERG), in a concentration-dependent manner, whereas D-arginine had no effect. L-Arg partially reversed the L-NA-induced inhibitory effect, and L-NA prevented the L-Arg-induced attenuation of the CAPs. Sodium nitroprusside reduced both the CAPs and the ERG, whilst potassium ferricyanide did not affect the CAPs, and potentiated the ERG. We conclude that, independent of the vasculature, endogenous NO modulates neurotransmission (i.e. light-evoked CAPs), but probably not phototransduction (i.e. light-evoked ERG) in this intact in vitro central nervous system preparation.

Effects of acrylamide on cotransmission in perivascular sympathetic and sensory nerves.

The effects of chronic administration of acrylamide on sympathetic and sensory nerves were examined in the mesenteric artery of rabbits. The noradrenaline (NA) content of the artery was significantly decreased and the total contractile response to electrical field stimulation (4-64 Hz) markedly reduced in the acrylamide group. This was not due to an impairment of the contractility of the smooth muscle or to alterations in the postjunctional receptors. At 16 Hz, only the purinergic component of sympathetic cotransmission was significantly reduced by acrylamide. At 64 Hz, both the purinergic and the adrenergic components were significantly decreased. Field stimulation of the artery pretreated with guanethidine and precontracted with NA produced a frequency-dependent relaxation which was prevented by capsaicin and thus mediated by perivascular sensory nerves. In contrast to its effects on sympathetic cotransmission, acrylamide resulted in a trend, although not significant, towards increased responses at each frequency studied (2-16 Hz). 2-Methylthio-ATP (2Me-S-ATP) caused significantly greater relaxation following acrylamide treatment while vasodilator responses to calcitonin gene-related peptide and substance P were unchanged. It is concluded that, in addition to its known action in producing neuropathy in myelinated somatic motor and sensory nerves, acrylamide causes damage to unmyelinated perivascular sympathetic fibres. Purinergic mechanisms may be particularly susceptible to acrylamide since both the purinergic component of sympathetic vasoconstriction and the relaxation in response to 2Me-S-ATP were affected by acrylamide treatment.

Cerebral blood flow changes during cortical spreading depression are not altered by inhibition of nitric oxide synthesis.

CBF increases concomitantly with cortical spreading depression (CSD). We tested the hypothesis that CBF changes during CSD are mediated by nitric oxide (NO). Male Wistar rats (n = 23) were subjected to KCl-induced CSD before and after administration of nitric oxide synthase (NOS) inhibitors N-nitro-L-arginine (L-NNA) or N-nitro-L-arginine methyl ester (L-NAME) and in nontreated animals. CBF, CSD, and mean arterial blood pressure were recorded. Brain NOS activity was measured in vitro in control, L-NNA, and L-NAME-treated rats by the conversion of [3H]arginine to [3H]citrulline. Our data show that the NOS inhibitors did not significantly change regional CBF (rCBF) during CSD, even though cortical NOS activity was profoundly depressed and systemic arterial blood pressure was significantly increased. Our data suggest that rCBF during CSD in rats is not regulated by NO.

L-NNA decreases cortical hyperemia and brain cGMP levels following CO2 inhalation in Sprague-Dawley rats.

The role of nitric oxide (NO) in the response to 5% CO2 inhalation was investigated by measuring 1) regional cerebral blood flow (rCBF) by laser-Doppler flowmetry and pial vessel diameter through a closed cranial window after topical NG-nitro-L-arginine (L-NNA, 1 mM), and 2) the time-dependent changes in brain guanosine 3',5'-cyclic monophosphate (cGMP) levels after L-NNA (10 mg/kg ip). When L-NNA (but not NG-nitro-D-arginine) was applied topically for 30 or 60 min, the response to hypercapnia was significantly attenuated. A correlation was found between inhibition of brain NO synthase (NOS) activity and the rCBF response (r = 0.77; P < 0.01). However, L-NNA applied 15 min before hypercapnia did not attenuate the increase in rCBF but did attenuate the dilation to topical acetylcholine. Inhalation of CO2 (5%) elevated brain cGMP levels by 20-25%, and L-NNA reduced this response. These data from the rat suggest that 1) a product of NOS activity is associated with hypercapnic hyperemia and the attendant increase in brain cGMP levels, and 2) hypercapnic blood flow changes may not be dependent on endothelial NOS activity within pial vessels.