ORIGINAL ARTICLE
Inhibition of nitric oxide production during
global ischemia ameliorates ischemic damage ofpyramidal neurons in the hippocampus
Takahiro Sasaki, Junnichi Hamada, Mamoru Shibata, Nobuo Araki and Yasuo Fukuuchi
Department of Neurology, School of Medicine, Keio University, Tokyo, Japan(Received for publication on April 2, 2001)

Abstract. We examined the relationship between nitric oxide (NO) production and delayed neuronal
death (DND), in the rat hippocampus induced by 21 minutes of transient global ischemia produced by
the occlusion of both of the common carotid arteries combined with systemic hypotension. NO production during ischemia and reperfusion was investigated by quantifying the nitrite (NO2 C ) levels of
the in vivo microdialysis samples collected every 3 minutes from the hippocampus. To determine theorigin of NO production, we studied the effects of the focal administration of NG -nitro-L-arginine
methyl ester (L-NAME), an inhibitor of the constitutive NO synthase (NOS). We also carried out
systemic administration of a selective neuronal NOS inhibitor, 7-nitroindazole (7-NI). Rats weregrouped as follows: group 1 (n F 22), vehicle; group 2 (n F 19), L-NAME; group 3 (n F 12), 7-NI; andgroup 4 (n F 12), a sham operation. The role of NO in the hippocampal DND was investigated histologically one week after ischemia. The level of NO production was signi®cantly decreased in groups 2
and 3 as compared to group 1 in which NO production was signi®cantly increased (p H 0:05). The
density of remaining neurons in the CA1 area was signi®cantly reduced only in group 1 (p H 0:01).
Taken together, it can be concluded that NO production by neuronal NOS during ischemia andreperfusion resulted in DND in the CA1 region of the rat hippocampus. (Keio J Med 50 (3): 182±187,
September 2001)
Key words: in vivo microdialysis, delayed neuronal death, NG -nitro-L-arginine methyl ester, 7nitroindazole, rat

Introduction
The hippocampus is known to be one of the brain
areas ``vulnerable'' to an ischemic insult.1 Some clinicalcases of the global ischemia due to transient systemic
hypotension, in which neurological de®cits such as amnesia often develop as sequelae, have been reported tohave pathological lesions restricted to hippocampal
neurons.2,3 Some form of vascular dementia may be
attributed to the vulnerability of the hippocampus toischemia. The pathophysiological change corresponding
to these cases in animal models is thought to be delayed
neuronal death (DND), which is slowly progressive cell
death originally found in the CA1 sector of the gerbil
hippocampus after transient global ischemia.4 Although
this selective vulnerability of the CA1 sector of the

hippocampus to cerebral ischemia is well known, themechanism underlying this vulnerability has not yet
been elucidated.
The glutamate-calcium theory5,6 is one of the mostwell known theories that can explain neuronal death,
including the mechanism of DND not directly mediatedby energy failure. During ischemia, subsequent activation of NMDA-type receptors induces a massive in¯ux
of Ca 2 into neurons. Secondary to the elevation of
intracellular calcium, constitutive nitric oxide synthase(NOS) is activated by calmodulin and results in NO
production.
The participation of NO in DND has not been demonstrated. Elucidating the mechanism of slowly progressive neuronal death, including DND, is important,since it concerns the treatment of not only global

Reprint request to: Dr. Takahiro Sasaki, Department of Neurology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku,
Tokyo, 160-8582, Japan, e-mail: tsasaki@med.keio.ac.jp
182Keio J Med 2001; 50 (3): 182±187

ischemia but also the ``penumbra'' around the ischemic
core. However, few studies have been done on the direct relation between the NO production and DND.7±9
The purpose of this study is to investigate the relationship between the NO production during cerebral
ischemia and reperfusion, and the histological changes
observed in the rat hippocampus one week after transient global ischemia.
Materials and Methods
Male Sprague-Dawley rats weighing 250±350 g wereanesthetized by an intraperitoneal injection of pentobarbital sodium (40 mg/kg). The body temperature wascontinuously monitored and maintained at 37.0±37.5 C
during ischemia and reperfusion with a thermostaticallycontrolled heating pad connected to a rectal thermistor
probe. The unilateral femoral vein and a tail artery
were catheterized for induction of exsanguination andcontinuous monitoring of the blood pressure respectively. The blood pressure was recorded continuously
using a multi-pen recorder via a pressure transducer.
Both of the common carotid arteries were isolated andloops made of PE-10 polyethylene catheter were passed
around the carotid arteries for subsequent occlusion.
Animals were placed in a stereotaxic frame with theskull level. Following skull exposure, a small burr hole
was drilled at 4.8 mm posterior and 5.3 mm lateral tothe bregma, bilaterally, allowing insertion of a doubleluminal type microdialysis probe (outer diameter 220
mm, length 3 mm, cellulose membrane) into the hippocampus so that the probe tip was positioned 4.8 mm
posterior, 5.3 mm lateral, and 7.5 mm ventral to thebregma. The microdialysis probe was perfused with
Ringer's solution (NaCl, 147 mM; KCl, 4 mM; andCaCl2 , 2.5 mM) at a constant rate of 2 ml/min employing
a microsyringe pump. After a 2-hour equilibrium period, the dialysate was collected every 3 min in polyethylene tubes kept at 15 C.
Physiological parameters (blood pressure, PaO2 ,PaCO2 , and the pH of arterial blood) were measured
before the induction of ischemia.
Global ischemia was induced by occlusion of bothcommon arteries using polyethylene loops with systemic hypotension. The mean arterial pressure wasmaintained below 50 mmHg10 by drawing blood fromthe femoral vein. The focal cerebral blood ¯ow wasmeasured by laser-Doppler ¯owmetry in several cases
to ensure that ischemia had been induced. After 21 minof ischemia, cerebral reperfusion was achieved byreleasing the polyethylene loops around the common
carotid arteries and normalizing the blood pressure.
The animals were decapitated 6 hours (n  4), 24 hours(n  4) and one week after ischemia (n  4), and thebrains were immediately immersed in 4% formalde-183

hyde. This ®xation method assured optimal visualization of the hippocampus, and did not affect the ischemic
damage grading used in this analysis. The brains were
excised 1 mm in front of the insertion sites of the
microdialysis probes and cut into 3 mm coronal slices.
Sections (10 mm) were cut from an area in close proximity to the probe insertion sites and stained with hematoxylin and eosin for ischemic grading and neuronalcounting.
For the in vivo microdialysis study, the animals
studied were divided into the following groups: Group 1(n  22): global ischemia and reperfusion were induced
as previously described. Group 2 (n  19): focal administration of 4 mM NG -nitro-L-arginine methyl ester(L-NAME), a constitutive NOS inhibitor, via a microdialysis probe was started 24 min before inducing
ischemia in the same manner as in group 1. L-NAME
was administered continuously for 60 min. Group 3(n  12): 7-nitroindazole (7-NI), a neuronal NOS inhibitor, was suspended in peanut oil by sonication. 7-NI
(30 mg/kg) was so insoluble in water that it had to beadministered intraperitoneally 30 min before inducing
ischemia in the same manner as in group 1. Group 4(n  12): a sham operation was performed. The unilateral femoral vein and a tail artery were catheterized.
Blood pressure was recorded continuously. Both common carotid arteries were isolated and loops made of
PE-10 polyethylene catheter were passed around the
carotid arteries, but carotid artery occlusion was not
performed.
NO2 ÿ levels in dialysate samples were measured by
the Griess reaction.11 Brie¯y, samples were injected
into a ¯ow-through system and delivered by a carrier
solution containing 1% NH4 Cl at 10 ml/min, reaching a
mixing tee where the samples react with the Griess
agents (one part 1% sulfanilamide and one part 0.1%
N-(naphthyl)-ethylenediamine) to yield an azo dye
after incubation at 40 C. Subsequently, the samples
underwent spectrophotometric analysis to determine
NO2 ÿ levels. We have already demonstrated theabsorbance at 546 nm to be proportional to NO2 ÿ levels
in our system.11 In the statistical analyses, the data
were expressed as a percentage of the baseline level,
the mean value of the preceding three samples before
ischemia and reperfusion.
The grading scale developed for this study took into
account the severity of the ischemic damage. The nucleus of an ischemic neuron deteriorates in stages thatcan be readily assessed by microscopic observation at
320 magni®cation in hematoxylin-eosin stained 10 mm
plastic sections.
The following histological changes were graded into
three categories as reported by Eke et al.:12 histologically normal, mild ischemic changes (e.g., smeared
nuclear-cytoplasmic border, mild nuclear shrinkage,

184

Sasaki T, et al: NO production by neuronal NOS exerts delayed neuronal death

Table 1 Physiological parameters were measured before the induction of ischemia. There were no signi®cant differences among thephysiological parameters in the four groups studiedGroup 1 (n  22)
Group 2 (n  19)
Group 3 (n  12)Group 4 (n  12)pH

PO2 (mmHg)

PCO2 (mmHg)

7:42 G 0:36
7:43 G 0:16
7:41 G 0:03
7:44 G 0:36

84:1 G 6:4
85:1 G 3:7
82:6 G 8:5
82:5 G 7:4

39:8 G 6:8
41:1 G 5:8
38:4 G 6:2
38:8 G 6:9

and mild cytoplasmic eosinophilia), and severe ischemicchanges (e.g., a triangular shrunken neuron lacking
nuclear-cytoplasmic detail, and strong eosinophilicchanges in the cytoplasm). Neurons with recognizable
nuclei were counted in each area of the hippocampus(e.g., CA1, CA2, and CA3) and classi®ed into theischemic categories described above. The degree ofischemic change in each area of the hippocampus was
expressed as the density of surviving neurons and as thepercentage of each category. The mean and standard
deviations for the degree of ischemic change in all areas
are depicted for each group. The sections were gradedin a double-blinded manner by a neuropathologist who
was blind to the identi®cation of the groups.
The ANOVA multiple measurement and MannWhitney test were used for the microdialysis and histological studies, respectively.
The Experimental Committee of Keio University
approved the experimental protocol as meeting theExperimental Animal Guidelines of Keio University
School of Medicine.
Results
There were no signi®cant differences in physiologicalparameters among the four groups studied (Table 1). In
group 1, global ischemia gave rise to a signi®cant increase in NO2 ÿ 15, 18 and 21 min after the onset ofischemia and 6, 9, 12 and 15 min after reperfusion, as
compared to group 4. In group 2, administration of LNAME induced a signi®cant decrease in NO2 ÿ 18 minafter the onset of ischemia and 12 min after reperfusion,as compared to group 1. In group 3, 7-NI also induced asigni®cant decrease 18 and 21 min after the onset ofischemia and 9, 12 and 15 min after reperfusion, as
compared to group 1 (Fig. 1). These data suggest that
production of NO through constitutive NOS, mainly
neuronal NOS, increases during ischemia and reperfusion.
The temporal pro®le of neuronal death in the hippocampal CA1 area was studied. Although the densities of neurons in CA1 six hours and 24 hours after
ischemic insult did not differ signi®cantly from those of
the control group, that of CA1 neurons one week afterFig. 1 NO2 ÿ levels in dialysate samples, collected by in vivo microdialysis technique, were measured by the Griess reaction. In group
1(9), global ischemia gave rise to a signi®cant increase in NO2 ÿ 15,
18 and 21 min after the onset of ischemia and 6, 9, 12 and 15 min after
reperfusion compared to group 4(v) (*p < 0:05, **p < 0:01). In
group 2(h), administration of L-NAME induced a signi®cant decrease in NO2 ÿ 18 min after onset of ischemia and 12 min after
reperfusion compared to group 1 (y p < 0:05). In group 3(y), 7-NI
also induced a signi®cant decrease 18 and 21 min after onset ofischemia and 9, 12 and 15 min after reperfusion compared to group 1(] p < 0:05, ]] p < 0:01). ANOVA multiple measurement. Values aremean and standard deviation.

ischemia was signi®cantly decreased as compared withthe other groups. (Fig. 2) These data suggest that the
global ischemia employed in the present study induced
DND.
By histological examination, the numbers of remaining neurons in CA1 in groups 2, 3 and 4 signi®cantly
exceeded that in group 1 (Fig. 3). There were no signi®cant differences between CA2 and CA3 areas (datanot shown). The ratio of normal neurons in CA1 ingroup 3 was also signi®cantly higher than that in group
1 (Fig. 4). There were no signi®cant differences between the CA2 and CA3 areas. These data suggest that
inhibition of constitutive NOS, particularly neuronal
NOS, has a protective effect against neuronal damage
in the CA1 region one week after transient global
ischemia.
Discussion
Our present data suggest a causal relationship between NO production by neuronal NOS, during andshortly after ischemia, and neuronal death of pyramidal
neurons in the CA1 region of the rat hippocampus one
week after ischemia. Although Lei et al.7 reported acontinuous temporal pro®le of NO production during
global ischemia and reperfusion, and the protective

Keio J Med 2001; 50 (3): 182±187

185Fig. 4 The histological changes, estimated from hematoxylin/eosinstained section one week after transient global ischemia, were graded
into three categories. The ratio of normal neurons in CA1 in group 3
was signi®cantly higher than that in group 1. S: severe ischemicchange, M: moderate ischemic change, N: normal. *p < 0:05, MannWhitney's U test. Values are mean G SD.

Fig. 2 Temporal pro®le of neuronal death on hippocampus CA1 wascon®rmed. A, coronal brain sections (10 mm) of the CA1 sector ofhippocampus of animals killed at indicated time points after global
ischemia were stained with hematoxylin and eosin. Scale bar, 50 mm.
B, The density of neurons in CA1 six hours and twenty-four hours
after ischemic insult and also treated with 7-NI was not signi®cantly
different from that of control. However, the density of neurons in
CA1 one week after ischemia was signi®cantly decreased against
other. *p < 0:05, Mann-Whitney's U test. Values are mean G SD.

Fig. 3 The density of neurons in hippocampus CA1 was estimatedfrom hematoxylin/eosin-stained section one week after transient
global ischemia. The density in group 2, group 3 and group 4 was signi®cantly larger than that in group 1. *p < 0:01, Mann-Whitney's U
test. Values are mean G SD.

effect of 7-NI against DND in the gerbil hippocampus,
no data from rats have as yet been reported. Differences among species are very important when subtle
changes, such as measuring small quantities of nitrite
and distinguishing between protective and harmful
effects of NOS, are considered.
In the present study, 21 min global ischemia
increased the level of NO production in the hippocampus although a previous study reported a decreasein that of NO production during ischemia.7 This increase in NO production was seen during both thelatter half of the ischemic period and the reperfusion
period. The lack of an NO production increase justafter the onset of ischemia may be explained by thevery short half-life (less than a few seconds) of NO. NO,
an unstable molecule with unpaired electrons, can combine with several other molecules and is metabolized
immediately after its production. The hypoxic state
during ischemia is likely to cause some delay in NO2 ÿ
formation, since the rate of NO oxidation is criticallydependent upon the oxygen concentration.13 Enhancement of NO production during ischemia must be transient, since the supply of oxygen or L-arginine, both ofwhich are required for NO production, gradually decreases with the progression of ischemia. Besides,
NOS itself may degenerate in response to an ischemic
insult. The total amount of NO production during theischemic period may decrease even if NO production
increases transiently during the ischemic period. Thus,
to evaluate the production of NO precisely during
ischemia, NO should be monitored continuously withmeasurements taken at short intervals as in our study.
The production of NO also increased during the reper-

186

Sasaki T, et al: NO production by neuronal NOS exerts delayed neuronal deathfusion period and the amount of NO was higher thanthat in the ischemic period in the present study. This
may result from an increased supply of the NOS substrate, L-arginine, and oxygen due to reperfusion. Although we did not measure NO itself but rather its
product, NO2 ÿ , the measurement of NO2 ÿ alone is suf®cient to evaluate the NOS activity in the brain. Nitrate(NO3 ÿ ) measurement appears to be unnecessary, since(1) the nitrogen atoms in NO2 ÿ and NO3 ÿ are derived
exclusively from one of the two nitrogens in arginine14
and (2) NO2 ÿ is not converted to NO3 ÿ 15 in the brain
tissue during the early phase of ischemia.16 The rationale for measuring NO2 ÿ in this model has been discussed in detail elsewhere.17
In the present study, administration of L-NAME
during and shortly after ischemia exerted a neuroprotective effect in the hippocampal CA1 region one
week after ischemia. The effect of systemic administration of L-NAME on ischemia is controversial. Sancesario, et al.18 reported that intraperitoneal administration of L-NAME (50 mg/kg) every other day that was
initiated four days before 10-minute ischemia inducedby common carotid artery occlusion (CCAO), resulted
in a greater ischemic damage to CA1 neurons ®ve days
after ischemia.18 On the other hand, Shapira et al.
showed that intraperitoneal administration of the constitutive NOS inhibitor, NG -nitro-L-arginine (L-NA)(5mg/kg), 4 hours before 5-minute ischemia induced by
CCAO, would suppress ischemic changes in CA1 neurons six days after ischemia.19 Stronsznajder et al.20 and
Kohno et al.8 also suggested that constitutive NOS
inhibitors had a neuroprotective effect one-week after a
5-minute ischemic insult. These discrepancies may be
due to the amount of the NOS inhibitor used, that is, alarge amount of the NOS inhibitor (10±50 mg/kg)
would exaggerate the ischemic damage to CA1 neurons. We speculate that small amounts of constitutive
NOS inhibitors can suppress neuronal NOS, thereby
apparently exerting neuroprotective effects. However, alarge amount of a constitutive NOS inhibitor may also
suppress endothelial NOS, possibly producing a neurotoxic effect. In our study, 4 mM L-NAME was focallyadministered via a microdialysis probe and a neuroprotective effect was observed. L-NAME is so lipidsoluble that it can readily penetrate neurons near the
microdialysis probe to attenuate neuronal NOS activity.
To con®rm the above assumption, we further investigated the effect of 7-NI. 7-NI, a selective neuronal
NOS inhibitor, was administered intraperitoneally inthe same model, which demonstrated a neuroprotective
effect on the outcome of ischemic neuronal damage at
one week after transient ischemia. This result suggeststhat the neuronal NOS activity in the hippocampus isneurotoxic, that is, neuronal NOS may play a neurotoxic role in delayed neuronal death as described inother studies.9 To investigate neuronal death resulting
from ischemia, the in¯uence of hypothermia should beconsidered, since hypothermia is known to be neuroprotective.21 In the present study, however, hypothermia exerted no neuroprotective effects. The effectof inducible NOS should also be considered. However,
the effects of focally administering L-NAME and 7-NI
persisted for about one day22 and inducible NOS activity peaked two days after transient ischemia. Therefore,
it is probable that L-NAME or 7-NI itself had no directin¯uence on the activity of inducible NOS in our study.
All three groups were equally in¯uenced by inducible
NOS or the one week process of maturation of neuronal death in our study. Thus, it can be concluded thatsuppression of neuronal NOS activity during ischemia
and reperfusion exerted protective effects against
DND.
NO production triggers several events during ischemia and early phase reperfusion, including depletion of
DNA by peroxynitrite (ONOO ÿ ),23 inhibition of superoxide dismutase in mitochondria,24 reversible suppression of mitochondrial oxidation via competition withoxygen,25 and suppression of GAPDH activity resulting
in exhaustion of ATP.26 Fragmentation of DNA, whichis due to the depletion of nuclear DNA, activates poly
ADP-ribose polymerase (PARP) in the nucleus. As
PARP is an enzyme which adds ADP-ribose from NAD
to protein in the nucleus, DNA damage activates PARP
to add a large amount of ADP-ribose to histone and
PARP itself. Each addition of ADP-ribose consumes
one molecule of NAD which is synthesized by fourmolecules of ATP. Damage to mitochondrial enzymes
by NO also leads to exhaustion of NAD and ATP,
resulting in energy failure and neuronal death.27 The
suppression of NO production during ischemia and theearly phase of reperfusion might attenuate the pathological mechanisms described above and signi®cantly
diminish neuronal death during the ®rst week after
ischemia.
Our experiment demonstrated a causal relationshipbetween NO production by neuronal NOS, during andshortly after ischemia, and neuronal death of pyramidal
neurons in the CA1 region of the rat hippocampus one
week after ischemia. These data suggest that NO synthesized by neuronal NOS promotes DND in rats.
References
1.

Pulsinelli WA: Selective neuronal vulnerability: morphologicaland molecular characterictics. In: Kogure K, Progress in Brain
Research V63, Molecular Mechanisms of Ischemic Brain Damage, Amsterdam, Elsevier, 1985; 29±372. Zola-Morgan S, Squire LR, Amaral DG: Human amnesia andthe medial temporal region: enduring memory impairment following a bilateral lesion limited to ®eld CA1 of the hippocampus. J Neurosci 1986; 6: 2950±2967

Keio J Med 2001; 50 (3): 182±187
3.
4.
5.
6.
7.

8.

9.

10.

11.

12.
13.
14.
15.
16.

Petito CK, Feldmann E, Pulsinelli WA, Plum F: Delayed hippocampal damage in humans following cardiorespiratory arrest.
Neurology 1987; 37: 1281±1286
Kirino T: Delayed neuronal death in the gerbil hippocampus
following ischemia. Brain Res 1982; 239: 57±69
Choi DW: Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 1995; 18: 58±60
Rothman SM, Olney JW: Excitotoxicity and the NMDA receptor ± still lethal after eight years. Trends Neurosci 1995; 18: 57±
58
Lei B, Adachi N, Nagaro T, Arai T, Koehler RC: Nitric oxide
production in the CA1 ®eld of the gerbil hippocampus after
transient forebrain ischemia: effects of 7-nitroindazole and NGnitro-L-arginine methyl ester. Stroke 1999; 30: 669±677
Kohno K, Ohta S, Furuta S, Kohno K, Kumon Y, Sakaki S:
Intraventricular administration of nitric oxide synthase inhibitors
prevents delayed neuronal death in gerbil hippocampal CA1
neurons. Neurosci Lett 1995; 199: 65±68
Nanri K, Montecot C, Springhetti V, Seylaz J, Pinard E:
The selective inhibitor of neuronal nitric oxide synthase, 7nitroindazole, reduces the delayed neuronal damage due toforebrain ischemia in rats. Stroke 1998; 29: 1248±1253
Smith ML, Bendek G, Dahlgren N, Rosen I, Wieloch T, Siesjo
BK: Models for studying long-term recovery following forebrainischemia in the rat. 2. A 2-vessel occlusion model. Acta Neurol
Scand 1984; 69: 385±401
Ohta K, Araki N, Shibata M, Hamada J, Komatsumoto S, Shimazu K, Fukuuchi Y: A novel in vivo assay system for consecutive measurement of brain nitric oxide production combinedwith the microdialysis technique. Neurosci Lett 1994; 176: 165±
168
Eke A, Conger KA, Anderson M, Garcia JH: Histologic assessment of neurons in rat models of cerebral ischemia. Stroke 1990;
21: 299±304
Beckman JS, Koppenol WH: Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996; 271:
C1424±C1437
Iyengar R, Stuehr DJ, Marletta MA: Macrophage synthesis ofnitrite, nitrate, and N-nitrosamines: precursors and role of therespiratory burst. Proc Natl Acad Sci U S A 1987; 84: 6369±6373
Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS: Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 1988; 27: 8706±8711
Ignarro LJ, Fukuto JM, Griscavage JM, Rogers NE, Byrns RE:

187

17.
18.

19.
20.
21.

22.

23.

24.

25.
26.

27.

Oxidation of nitric oxide in aqueous solution to nitrite but notnitrate: comparison with enzymatically formed nitric oxide from
L-arginine. Proc Natl Acad Sci U S A 1993; 90: 8103±8107
Shibata M, Araki N, Hamada J, Sasaki T, Shimazu K, Fukuuchi
Y: Brain nitrite production during global ischemia and reperfusion: an in vivo microdialysis study. Brain Res 1996; 734: 86±90
Sancesario G, Iannone M, Morello M, Nistico G, Bernardi G:
Nitric oxide inhibition aggravates ischemic damage of hippocampal but not of NADPH neurons in gerbils. Stroke 1994; 25:
436±43
Shapira S, Kadar T, Weissman BA: Dose-dependent effect ofnitric oxide synthase inhibition following transient forebrainischemia in gerbils. Brain Res 1994; 668: 80±84
Strosznajder J, Chalimoniuk M, Samochocki M, Gadamski R:
Nitric oxide: a potent mediator of glutamatergic neurotoxicity inbrain ischemia. Ann N Y Acad Sci 1994; 723: 429±432
Nurse S, Corbett D: Direct measurement of brain temperatureduring and after intraischemic hypothermia: correlation withbehavioral, physiological, and histological endpoints. J Neurosci
1994; 14: 7726±7734
Iadecola C, Xu X, Zhang F, Hu J, el-Fakahany EE: Prolonged
inhibition of brain nitric oxide synthase by short-term systemic
administration of nitro-L-arginine methyl ester. Neurochem Res
1994; 19: 501±505
Szabo C, Zingarelli B, O'Connor M, Salzman AL: DNA strand
breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite. Proc
Natl Acad Sci U S A 1996; 93: 1753±1758
MacMillan-Crow LA, Crow JP, Kerby JD, Beckman JS,
Thompson JA: Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts.
Proc Natl Acad Sci U S A 1996; 93: 11853±11858
Brown GC: Nitric oxide regulates mitochondrial respiration andcell functions by inhibiting cytochrome oxidase. FEBS Lett 1995;
369: 136±139
Dimmeler S, Ankarcrona M, Nicotera P, Brune B: Exogenous
nitric oxide (NO) generation or IL-1 beta-induced intracellular
NO production stimulates inhibitory auto-ADP-ribosylation ofglyceraldehyde-3-phosphate dehydrogenase in RINm5F cells. J
Immunol 1993; 150: 2964±2971
Zhang J, Dawson VL, Dawson TM, Snyder SH: Nitric oxide
activation of poly(ADP-ribose) synthetase in neurotoxicity.
Science 1994; 263: 687±689