l -Arginine ameliorates cerebral blood flow and metabolism and decreases infarct volume in rats with cerebral ischemia

BRAIN RESEARCH ELSEVIER

Brain Research 699 (1995) 208-213

Research report

L-Arginine ameliorates cerebral blood flow and metabolism and decreases infarct volume in rats with cerebral ischemia Zhen He, Setsuro Ibayashi *, Tetsuhiko Nagao, Kenichiro Fujii, Seizo Sadoshima, Masatoshi Fujishima Second Department of lnternal Medicine, Faculty of Medicine, Kyushu University,Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan

Accepted 6 July 1995

Abstract Effects of L-arginine, 300 mg/kg, i.p., on the regional cerebral blood flow (rCBF), brain metabolism, and infarct volume were examined in spontaneously hypertensive rats subjected to occlusion of both left middle cerebral artery and left common carotid artery. Rats treated with L-arginine had higher rCBF, determined by hydrogen clearance method, in the ischemic core (7 ± 1 ml/100 g/min, mean ± S.E.M.) and penumbral regions (16 + 2) than did rats treated with saline (5 ± 0 and 7 + 1, respectively). Simultaneously, L-arginine attenuated metabolic derangement in the ischemic tissue at 60 min, i.e. well maintained adenosine triphosphate (ATP) in ischemic region (1.29 + 0.07 mmol/kg in L-arginine group vs. 1.05 + 0.06 in saline group), and also close to normal levels in ATP (2.61 ± 0.02 mmol/kg vs. 2.45 + 0.05), glucose (2.29 ___0.12 mmol/kg vs. 1.80 ___0.17) and lactate (1.63 ±0.10 mmol/kg vs. 2.24 ___0.21) in periischemic region. In another experiment, the effects of L-arginine on rCBF in the subcortical regions and on infarct volume were evaluated. L-arginine, compared with saline, increased rCBF by 8 ml/100 g/min in the ischemic side and reduced infarct volume by 29% at 24 h of ischemia. These findings support that L-arginine may be potentially useful for the treatment of acute cerebral ischemia. Keywords: L-Arginine;Cortical ischemia; Cerebral blood flow; Brain metabolite; Infarct volume

1. Introduction Nitric oxide (NO) is a major candidate for endothelium derived relaxing factor. L-Arginine, as a well known precursor of NO, has been reported to increase regional cerebral blood flow (rCBF) and to decrease infarct volume in experimental cerebral ischemia in rats [14,16]. Furthermore, stimulation of fastigial nucleus causes focal production of NO associated with both increase in rCBF and attenuation of neuronal damage during focal cerebral ischemia [23]. In contrast to these beneficial effects of NO on cerebral circulation, overproduction of NO may be involved in aggravation of some pathological conditions such as epilepsy and cerebral infarction [5,13,18]. Thus, the role of NO may be a double-edged knife in cerebral ischemia. In this study, we produced focal cerebral ischemia by occlusion of both left middle cerebral artery (MCA) and left common carotid artery (CCA) in spontaneously hyper-

* Corresponding author. Fax: (81) (92) 632-2551. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)00907-8

tensive rats (SHR). We examined whether administration of L-arginine better preserved rCBF and metabolism on the ischemic hemisphere, especially in the ischemic core and penumbra. We also measured the volume of infarct and elucidated whether production of NO after L-arginine contributed to reduce the focal infarct volume.

2. Materials and methods Forty three male SHR ( 6 - 1 0 months old, weighing 320-435 g) were anesthetized with amobarbital (100 m g / k g b.wt., i.p.). A femoral artery was cannulated for recording mean arterial blood pressure (MABP) and for sampling blood for the measurements of blood gases, blood glucose and hematocrit. One femoral vein was cannulated for the administration of L-arginine or saline. Animals were allowed to spontaneously breath the room air and rectal temperature was kept at 37°C by a heating pad. Focal cerebral ischemia was induced by tandem occlusion of left distal M C A and ipsilateral C C A according to the method described by Brint et al. [2]. We divided rats

209

Z. He et al. / Brain Research 699 (1995) 208-213

SL

C

~

p

SR

NI

Left~~ ~'-

Right

I I lsehemic Side'4----I-------t~Nonisehemic Side I Fig. 1. Schematic illustration for five electrodes for regional cerebral blood flow and for three brain dissectingregions for mctaholitcanalysis. C = ischemic core; SL = subcortical region on the left ischemic side; P = penumbral region; SR= subcortical region on the right nonischemic side; NI = non-ischemiccortex. Regions 1 and 2 were dissected from the ischemic hemisphere and region 3 from the contralateral nonischemic hemisphere. into two groups, one for measurement of rCBF and metabolites (Group 1) and another for measurement of rCBF and infarct volume (Group 2). 2.1. G r o u p 1

Twenty-two rats were used to measure cortical rCBF (the ipsilateral ischemic core and the penumbral or alternatively, the contralateral non-.ischemic cortical regions; Fig. 1) and brain tissue metabolites (lactate, pyruvate, ATP and glucose) after 1 h of focal cerebral ischemia. One group, namely arginine group (A-group; 11 rats), received L arginine (300 m g / k g , i.p.) and the other group, as vehicle group (V-group; 11 rats), received saline alone (3 m l / k g , i.p.). Either L-arginine or saline was administered 20 min before the induction of cerebral ischemia.

2.2. G r o u p 2

In 21 rats, subcortical rCBF in bilateral hemisphere (Fig. 1) was measured during the first 2 h of focal cerebral ischemia. Then, the infarct volume was determined at 24 h of cerebral ischemia. In 9 rats, L-arginine was administered (300 m g / k g , i.p.) 20 min before cerebral ischemia and an additional 200 m g / k g , i.v., was given at 1 h after the ischemia (A-group). Another 12 rats received 3 m l / k g of saline as a vehicle (V-group). Hydrogen clearance method was used for rCBF determination [1,22]. In Group 1, the teflon-coated platinum electrodes were placed in the ipsilateral ischemic core region (5 m m lateral to the bregma), penumbral region (2.5 m m lateral and 1 mm posterior to the bregma) and the contralateral non-ischemic region (5 m m lateral to the bregma, Fig. 1). In Group 2, electrodes were put in the depth of 4 m m to the both sides of subcortical regions (3 m m lateral and 0.5 m m posterior to bregma; Fig. 1). Then, rCBF was recorded at baseline, 15 min after administration of L-arginine or saline, at 30 and 60 min of the cerebral ischemia in all animals (Groups 1 and 2), and at 90 and 120 min of ischemia in Group 2. The baseline rCBF was measured twice before the administration of drugs. For the metabolic study in Group 1, the brain was frozen in situ by liquid nitrogen at 60 min of ischemia and cut into 5 mm-thick slice [6] from 2 m m posterior to the frontal pole. Then, the slice was further separated into the ischemic cortical region (region 1), periischemic region (region 2) and non-ischemic cortical region (region 3) (Fig. 1). Lactate, pyruvate, ATP and glucose in each region were analyzed by a standard enzymatic method in a blinded fashion. In Group 2, the rats were decapitated at 24 h of cerebral ischemia. The seven 2-mm brain slices, started from the fontal pole, were stained by 1,3,5-triphenyltetrazolium chloride and the infarct area of every slice was calculated

Table 1 Arterial acid-base parameters, heraatocrit, and blood glucose before and after cortical cerebral ischemia At Rest Arginine(n = 20) Vehicle (n = 23) 60 rain Arginine(n = 11) Vehicle (n = 11) 120 min Arginine(n = 9) Vehicle (n = 12)

PaCO:.,(mmHg)

paO2 (mmHg)

pH

Hct (%)

BG (mg/dl)

46 + 1 46 + 1

83 + 1 87 + 2

7.40 ± 0.00 7.40 ± 0.00

45.4 + 0.5 45.4 + 0.4

106 + 3 103 + 3

39 ± 1 * 41 ± 1 t

96 ± 2 * 94 ± 1 *

7.42 ± 0.01 7.42 + 0.01 *

47.6 ± 0.6 * 47.4 + 0.0 *

109 + 7 110 ± 4

43 ± 1 * 42 ± 1 *

92 ± 1 * 92 ± 1 *

7.37 ± 0.01 * * * 7.44 ± 0.010 *

48.2 ± 1.0 * 48.6 + 0.7 *

109 + 5 * 123 +_3 *

Values are mean + S.E.M. At Rest = before cerebral ischemia;Arginine= argininegroup treated with L-arginine;Vehicle= vehicle group treated with saline; 60 min = 60 min after ischemia; 120 min = 120 rain after ischemia; Hct = hematocrit; BG = blood glucose. * P < 0.05, * * P < 0.01 vs. Vehicle. * p < 0.05 vs. At Rest in the same group.

Z. He et aL /Brain Research 699 (1995) 208-213

210

200"

by means of 'Epscan Mac 1.40' and 'NIH Image 1.54' computer programs. Values were compared by unpaired t-test between the two groups. Paired t-test was performed to compare the data of baseline with those after the ischemia. ANOVA was also used for comparison among the time changes of arterial blood parameters. All data were presented as mean ___S.E.M..

mmHg

160"

@ +

//

Arginine Vehicle

At After 30 60 90 Rest Drug -~------lschemia

3. Results Table 1 depicts arterial acid-base parameters, hematocrit and blood glucose in groups. During focal cerebral ischemia, all rats hyperventilated and paCO: significantly decreased and paOe increased. The changes in physiological variables in the two groups were similar but pH and blood glucose levels at 120 min were significantly higher in V-groups. The changes in systemic blood pressure are shown in Table 2 and Fig. 2. After administration of L-arginine, MABP decreased by 10-30 mmHg and soon returned to the baseline level. After the cerebral ischemia, MABP increased by 10-30 mmHg and MABP in V-group was significantly higher than that in A-group after 90 min. There were no significant differences in rCBF at rest in the five cerebral regions between the two groups (Table 2). After administration of L-arginine, rCBF increased in the core and penumbral regions ( P < 0.05, respectively), and

120

min

Fig. 2. M e a n arterial b l o o d pressure before and during 2 h o f t a n d e m occlusion of distal middle cerebral artery and ipsilateral carotid artery. B a r s represent S.E.M. A r g i n i n e = arginine g r o u p treated with L-arginine; Vehicle = vehicle g r o u p treated with saline; * P < 0.05 vs. Vehicle.

in the bilateral subcortical regions ( P = 0.13-0.16) as compared to the baseline values. In non-ischemic cortex, rCBF slightly increased in three of four rats following treatment of L-arginine, although no significant difference was detected. In rats receiving saline, the tandem occlusion of MCA and CCA induced much more profound reduction of rCBF in the core region, followed by in the penumbral and ipsilateral subcortical regions (Table 2 and Fig. 3). Administration of L-arginine significantly improved rCBF with increase of 2, 8 - 9 and 6-8 m l / 1 0 0 g / m i n in the core, penumbral and subcortical regions, respectively, in the ischemic hemisphere. Brain energy metabolites at 60 min of focal cerebral

Table 2 C h a n g e s in m e a n arterial blood pressure a n d cerebral b l o o d flow before and after cortical cerebral ischemia MABP

rCBF(ml/100 g/min)

(mmHg)

Conical

Subcortical

Core (n = 11)

P e n u m b r a l (n = 7)

N o n - i s c h e m i c ( n = 4)

Left (n = 9)

Right (n = 9)

156 ± 3 165 ± 5

54 ± 2 58 + 4 t

48 ± 3 61 __+7 -t

49 ± 6 46 ± 1

37+2 43±4

35±2 39±3

175+4 180±4 1 6 7 ± 5 ** 174 ± 4 *

7±15 7±1" * (n = 11)

17±2 * ~ 1 6 ± 2 ** * ( n = 6)

41±2 41±2 ( n = 5)

34±2 32+3 30±3 28±2

(n = 12)

(n = 12)

160 ± 2 160 ± 2

58 ± 3 56 ± 3

45 ± 4 46 ± 5

65 ± 10 63 ± 10

41±3 41±3

37±2 36±1

175±4 188 ± 4 189 ± 4 190 ± 5

5 + 1 :~ 5 ±0 * -

9±2 * 7± 1 * -

54±7 6 7 ± 11 -

28±2 ~ 24±2 ~ 22±25 20±2 s

41±2 40±3 41±2 39±2

Arginine Before ischemia: At Rest 15 min After ischemia: 30min 60min 90min 120 rain

* * * t

42+3 53+6 45+4 43+2

*t t t

Vehicle Before ischemia: A t Rest 15 min After ischemia: 30min 60min 90 min 120 min

Values are m e a n ± S.E.M. Arginine = arginine g r o u p treated with L-arginine; Vehicle = vehicle g r o u p treated with saline; 15 min = 15 min after L-arginine or saline treatment; M A B P = m e a n arterial blood pressure; r C B F = regional cerbral b l o o d flow. * P < 0.05, * * P < 0.01 vs. Vehicle; * p < 0.05, t p < 0.01 vs. At Rest.

Z. He et al. / Brain Research 699 (1995) 208-213

a

b

Arginine Vehicle

•

211

Core Arginine Vehicle lschemia Arginine N o n i s c h e m i a " " - 0 - - Vehicle

Penumbra

------O-- Atginine ~Vehicle - m O w Arginine Vehicle

---"-'O--

Nonischemia

%

%

50

200" 150

100

'

50

0

. At Rest

.

. After

.

'°t 0

30

60

rain

Drug ~1- Ischemia -I~

.

At

.

.

After

.

30

.

.

60

90

Ischemia

Rest Drug ~

120

rain

Fig. 3. Percent changes of regiona], cerebral blood flow in cortex (a) and in subcotex (b) after tandem occlusion of distal middle cerebral artery and ipsilateral carotid artery. Bars represent S.E.M. Arginine = arginine group treated with L-arginine; Vehicle = vehicle group treated with saline; * P < 0.05, • * P < 0.01 vs. Vehicle.

ischemia are shown in Fig. 4. Lactate level increased and ATP and glucose levels decreased in the ischemic cortical and periischemic regions in saline treated group. The

[] Arginine • Vehicle

mmol/kg Ischemic cortex

8 6 4 2 0

r*7 r-r-AT_

Periischemic region

r*7 7 ........

r*7

FT-

administration of L-arginine significantly inhibited to increase the lactate level in periischemic region (1.63 + 0.10 vs. 2.24 + 0.21 mmol/kg) and better preserved ATP in the ischemic cortical region (1.29+0.07 vs. 1.05 +0.06 mmol/kg) and the periischemic region (2.61 + 0.02 vs. 2.45 + 0.05 mmol/kg). The glucose level in the periischemic region was higher in A-group (2.29 + 0.12 mmol/kg) than that in V-group (1,80 + 0.17 mmol/kg). There were no differences in five metabolic parameters in non-ischemic regions between the two groups. The infarct volume in rats treated with L-arginine (Fig. 5) was 118 + 19 mm 3, which was 29% smaller than that in saline treated rats (168 + 14 mm 3, P < 0.05). The infarct area was also smaller in 5 - 7 slices, which were located at a

1. 0

mm 2

- - - - ' O F Arginine Vehicle

30

b

mm

3 20~

Nonischemic 3cortex 2!

0 Lactate

Pyruvate IJP Ratio (xl0) (/10)

ATP

Glucose

Fig. 4. Metabolic changes in the ischemic cortex (region 1 as shown in Fig. 1), periischemic region (regio:a 2) and non-ischemic cortex (region 3) after 60 rain of ischemia. Bars represent S.E.M. Arginine = arginine group treated with L-arginine; Vehicle = vehicle group treated with saline; L / P ratio =lactate/pyruvate ratio (without unit); ATP =adenosine triphosphate; ~P <0.01 vs. non-ischemic region in the same group; * P < 0.05, * P < 0.01 vs. Vehicle in the same region.

2

4

6

8 l0 12 14 mm

Arginine Vehicle

Fig. 5. Infarct areas (a) on 2-mm-thick coronal slices, started from the frontal pole, and infarct volume (b) at 24 h of tandem occlusion of distal middle cerebral artery and ipsilateral carotid artery. Bars represent S.E.M. Arginine = arginine group treated with L-arginine; Vehicle = vehicle group treated with saline; * P < 0.05 vs. Vehicle.

212

Z. He et al. /Brain Research 699 (1995) 208-213

10-14 mm from the frontal pole, in L-arginine treated group as compared with saline treated group (P < 0.05, respectively).

4. Discussion

The present study demonstrated that L-arginine treatment preserved rCBF in both the ischemic core and the perifocal regions including cortical penumbral and periischemic subcortical regions. L-Arginine also attenuated metabolic derangement in the ischemic cortical region and periischemic region and reduced infarct volume by 29%. Therefore, production of NO may be beneficial to maintain rCBF and metabolism, and thus, favorable to acute ischemia. L-Arginine, a precursor of NO, effectively dilates pial arterioles and increases rCBF [16]. Infusion of 300 m g / k g L-arginine reportedly increases rCBF by 22-33% in the ischemic core in SHR, and by 25% in the dorsolateral cortex in normotensive rats [15,16]. Furthermore, infusion of NO donors, sodium nitroprusside or 3-morpholinosydnonimine increases rCBF by about two times of control in the ischemic penumbral region [24,25]. Our observations are consistent with these previous reports. In the present study, L-arginine increased rCBF to the penumbral area from 7-9 ml/100 g / m i n to 16-17 ml/100 g / m i n (Table 2). Several experimental studies have suggested that synaptic transmission is inhibited when rCBF is decreased below 15-18 ml/100 g / m i n and ion pumping failure occurs at level of rCBF below 10-12 ml/100 g/min. Thus, it is likely that this small but important increase in rCBF by L-arginine significantly protects cerebral tissue from being infarcted. During the cortical ischemia, the metabolic derangement in ischemic cortical regions was pronounced and the variables, such as lactate, pyruvate, ATP and glucose were parallel to those reported by Folbergrov~ and Nowicki et al. [6,17]. The administration of L-arginine inhibited the metabolic derangement in the ischemic cortical region, increasing ATP by approximately 7%, which was associated with increase of rCBF by 2 and 7-9 ml/100 g / m i n in ischemic core and penumbra regions, respectively. A slight ischemic metabolic changes were observed in the subcortical ischemic region in V-group but not in A-group (Fig. 4). During ischemic intervention, rCBF to dorsal part of the caudoputamen in V-group gradually decreased to 61.3% of the baseline (mean value 24 ml/100 g/min) (Table 2). The level of rCBF is close to a reported flow threshold of energy status (25 ml/100 g/min) [8,12] and may correspond with a mild metabolic derangement at 60 min of ischemia in V-group. Thus, it is conceivable that the administration of L-arginine attenuated ischemic metabolic changes in both of the cortical and subcortical areas by the improvement of rCBF to these regions, suggesting that NO-mediated tissue sparing effects are pre-

dominantly based on vasodilating action. In contrast, it is possible that the observed increase in rCBF occurred secondary to amelioration of the metabolic derangement by L-arginine. In the absence of L-arginine, a brain NO synthase can generate superoxide and hydrogen peroxide [7,11,20], which are known to be neurotoxic. One of the explanations for a possible metabolic improvement by L-arginine is that the pretreatment of L-arginine prevented NO substrate from exhaustion and attenuated the sequential production of these materials. Time-related changes of NO action, however, favor that the increase in flow primarily occurs. It has been demonstrated that L-arginine increases CBF minutes before recovery of the electroencephalographic amplitude in the ischemic penumbra, suggesting that the increase in blood flow occurs primarily and is not related to the increased neuronal activity [4]. The most likely site for the conversion of L-arginine into NO is in cerebral blood vessels rather than in neurons because L-arginine-induced pial dilation can be blocked by topical nitro-L-arginine methyl ester at concentrations and time intervals that do not reduce the brain NO synthase activity [16]. Actually, production of NO may be beneficial in the first minutes or hours after the onset of ischemia but neurotoxic after many hours or days [19]. It has now been experimentally demonstrated that NO is detrimental during the late stages of cerebral ischemia. After focal ischemia there is an induction of inducible NO synthase that contributes to tissue damage [9,10]. Thus, a vasodilating action of NO predominates the neurotoxic effect during first several hours of ischemia. L-Arginine, in addition to being the precursor of NO, has also other biological effects that could potentially affect the outcome of the ischemic tissue. These include, for example, polyamine synthesis and release of growth hormone [21]. A possible role for NO, however, is suggested by the potency of the L-arginine but not the D-amino acid isomer and by the inhibition of the vasodilator response by nitro-L-arginine methyl ester [15]. Based on these findings, we consider that L-arginine induced amelioration of metabolic derangement followed by reduction of infarct volume is related to NO-mediated vasodilation. After occlusion of distal MCA, the ischemic area can be supplied via small diameter pial surface collaterals of the MCA anastomosing with rami of the anterior (ACA) or posterior (PCA) cerebral arteries. Numerous branches of the PCA join rami of the MCA but not the ACA, along a course roughly parallel to the transverse fissure separating occipital and cerebellar structure and the PCA-MCA anastomoses are intermediate in position between the dorsal ACA-MCA junction and the ventral anastomoses of the arterial circle of Willis [3]. In this experiment, L-arginine decreased infarct area limited in those slices far from frontal pole (Fig. 5), which is similar to the previous reports [15,25], suggesting that posterior circulation might mainly supply collateral blood flow to the ischemic area in SHR.

Z. He et al. / B r a i n Research 699 (1995) 208-213

In conclusion, we have .demonstrated that u-arginine decreased infarct volume following focal cerebral ischemia. It is considered that better preserved blood flow by L-arginine to the ischemic core as well as the perifocal (penumbral and subcortical) areas associated with reduced metabolic derangement may be primarily responsible for the protective effects of L-arginine against cerebral ischemia.

Acknowledgements We thank Miss Y. Sonoda and Miss M. Kumakawa for their technical assistance.

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