From the Departments of Pediatrics (S.A., B.T.), Anesthesiology (H.R.T.,
D.J.C.), and Physiology, Division of Perinatal Biology (W.J.P.), Loma Linda
University School of Medicine, Loma Linda, Calif.
Correspondence to Stephen Ashwal, MD, Department of Pediatrics, Loma Linda University School of Medicine, Coleman Pavillon, Loma Linda, CA 92354. E-mail Stephen_Ashwal{at}ccmail.llumc.edu
Methods—Based on previous studies that certain anatomically
well-defined areas are prone to become either core or penumbra after
middle cerebral artery occlusion (MCAO), we measured NOS activity in
these areas from the right and left hemispheres in a spontaneously
hypertensive rat filament model. Four groups were studied: (1) controls
(immediate decapitation); (2) 1.5 hours of MCAO with no reperfusion
(R0); (3) 1.5 hours of MCAO with 0.5 hour of reperfusion (R0.5);
and (4) 1.5 hours of MCAO with 24 hours of reperfusion
(R24). Three groups of corresponding isoflurane sham controls were also
included: 1.5 (S1.5) or 2 (S2.0) hours of anesthesia and
1.5 hours of anesthesia+24 hours of observation (S24).
Results—Control core NOS activity for combined right and left
hemispheres was 129% greater than penumbral NOS activity
(P<0.05). Combined core NOS activity was also greater
(P<0.05) in the three sham groups: 208%, 122%, and
161%, respectively. In the three MCAO groups, ischemic and
nonischemic core NOS remained higher than penumbral regions
(P<0.05). However, NOS activity was lower in the
ischemic than in the nonischemic core in all three
groups: R0 (29% lower), R0.5 (48%), and R24 (86%)
(P<0.05). Addition of cofactors (10 µmol/L
tetrahydrobiopterin, 3 µmol/L flavin adenine
dinucleotide, and 3 µmol/L flavin
mononucleotide) increased NOS activity in all groups and
lessened the decrease in ischemic core and penumbral NOS.
Conclusions—Greater NOS activity in core regions could explain in
part the increased vulnerability of that region to ischemia and
could theoretically contribute to the progression of the infarct over
time. The data also suggest that NOS activity during ischemia
and reperfusion could be influenced by the availability of cofactors.
To investigate this possibility, we have chosen areas of known
vulnerability to ischemia (ie, core and penumbra) and measured
NOS activity in these regions under control and ischemic
conditions. We used a well-established rodent filament model of
unilateral proximal MCAO.9 In this model, the
ischemic core first appears in the lateral striatum after
approximately 30 minutes (Figure 1
Initially we determined NOS activity from regions predesignated as
"core" and "penumbra" in control SHR who were immediately
decapitated. Since our earlier studies suggested that the major
neurotoxic effects of NOS-derived NO occur early during reperfusion
rather than ischemia16 and because others
have also shown fluctuations in whole brain NOS activity during
ischemia and reperfusion,18 19 we
determined NOS activity in three additional groups: (2) at the end of
1.5 hours of MCAO; (3) after 1.5 hours of MCAO and 0.5 hour of
reperfusion; and (4) after 1.5 hours of MCAO and 24 hours of
reperfusion. Our purpose was to determine whether differences in NOS
activity were present in the ischemic core and penumbra
compared with contralateral homologous regions and to determine to what
degree this activity changed during ischemia and early or late
reperfusion.
Because changes in NOS activity during ischemia or reperfusion
could be influenced by NOS cofactor
availability,20 21 we repeated the NOS assays in
separate homogenate samples for core and penumbral regions
from both hemispheres after adding the cofactors BH4, FAD, and FMN in
concentrations shown to generate maximum
activity.21 If cofactor addition restored NOS
activity to preischemic or near-preischemic
levels, then alterations in cofactor concentrations during
ischemia or reperfusion may be important in the
postischemic regulation of NOS activity.
Establishment of Reversible Focal Cerebral Ischemia
Focal ischemia was accomplished with the use of a filament
model of temporary unilateral proximal MCAO, as previously reported by
our laboratory,13 23 which was based on the
earlier studies of Tamura and coinvestigators.24
A 3- to 4-cm incision was made in the left cervical region, and the
common carotid artery was exposed and covered for 5 to 10 minutes with
0.375% bupivacaine. The external carotid artery and several of its
branches were ligated, and a length of suture was loosely passed under
the internal carotid artery distal to the bifurcation to control vessel
backflow. A bulldog vascular clamp was also placed on the common
carotid artery approximately 1 cm proximal to the bifurcation. A nylon
filament (No. 4) with a silicone-beaded tip (0.26 mm) was inserted
into the external carotid artery and advanced approximately 19 mm
or until resistance was felt. The filament was secured with 5–0 silk,
the clamp and suture were removed, the area was irrigated with lactated
Ringer's solution, and the skin was closed. Previous studies from our
laboratory in the adult SHR after 1.5 hours of occlusion and in the pup
SHR after 4 hours have shown a reproducible infarct involving the
striatum and overlying cortex.13 23
Depending on the experimental protocol, the rats were killed as
follows: (1) anesthetized and immediately decapitated; (2)
after 1.5 hours of MCAO (R0); (3) after 1.5 hours of MCAO and 0.5 hour
of reperfusion (R0.5); and (4) after 1.5 hours of
MCAO, filament removal, and 24 hours of recovery (R24). In the three
sham groups (S1.5, S2.0, and S24) that corresponded to the three
occlusion/reperfusion experimental groups, all procedures and time
periods were identical except for lack of filament insertion into the
external carotid artery.
Brain Sectioning for Measurement of NOS Activity and TTC
Staining
Regions from the right and left hemispheres of section 2 that
corresponded to the ischemic core and penumbra were dissected.
We initially identified the midline between the two hemispheres and
then made a longitudinal cut (from top to bottom) approximately 2
mm from the midline through each hemisphere. This was done to avoid
mesial hemispheric structures, which are supplied primarily by the
anterior cerebral artery. We then made a transverse diagonal cut at
approximately the "2 o'clock" position (as shown in Figure 1
We used the histochemical stain TTC to verify that an ischemic
insult had occurred and used only those animals with definite evidence
of ischemia for measurement of NOS
activity.16 25 26 27 SHR from the three MCAO groups
that did not have evidence of ischemic injury were not included
because sufficient control and sham groups were already being studied
and we did not want to include animals who may have had incomplete
ischemia from insufficient filament occlusion or an atypical
cerebrovascular collateral circulation. We included only animals that
had a defined area of ischemic injury that measured at least
10% of the hemispheric area. TTC, a mitochondrial stain, may delineate
an area of ischemic mitochondrial injury as early as 15 to 20
minutes after occlusion but usually is considered to more accurately
reflect ischemic injury at a minimum of 4, but preferably 24,
hours of occlusion.25 Within the first 4 to 6
hours after injury, the TTC method more accurately correlates with
other histopathological methods that measure infarct area when the
stain is perfused intra-arterially into brain tissue before
decapitation rather than when immersed after
removal.25 26 By 24 hours, no significant
differences between the immersion and perfusion TTC methods are
apparent.25 Because we used a "sandwich"
technique, in which TTC staining was performed on the two outer
sections with the middle section taken for NOS assay, the immersion
technique was used. Our immersion technique may have underestimated the
area of ischemia in the R0 and R0.5 groups but probably
accurately measured the area of ischemia in the R24
group.25 26 Thus, for the two early MCAO groups
(R0 and R0.5), we included those animals that had at least a 10% area
of pallor because earlier studies have shown that with the immersion
TTC technique one can detect approximately a 10% area of pallor after
1 to 2 hours of MCAO.26 Also, because of these
differences in interpreting the potential reversibility of the area of
ischemic mitochondrial injury, we did not attempt to correlate
the areas of early ischemic injury with measurements of NOS
activity.
Two brain sections from each animal (sections 1 and 3) were
immersed in TTC solution for 10 minutes and then stored in 15 mL of
10% buffered formalin. The two sections were photographed with color
slide film (Ektachrome, Tungsten 160 ASA) and analyzed with a
Drexel/DUMAS image processing system. The infarct area of the posterior
surface of section 1 and the anterior area of section 3 were averaged
to obtain an estimate of the percent area of ischemic injury
that corresponded to section 2. The effect of edema on these
measurements was corrected by utilizing the method of Swanson et
al.28
Measurement of Core and Penumbral NOS Activity
Each specimen was homogenized in 50 mmol/L HEPES with
1 mmol/L EDTA at pH 7.4, then centrifuged at
1500g for 10 minutes at 4°C. The supernatants were
analyzed for protein content with the use of the BCA Protein
Assay Reagent (Pierce) with bovine serum albumin as a standard
and then frozen at -80°C for subsequent measurement of NOS activity.
The ratio of tissue wet weight to homogenization
buffer volume was routinely adjusted for each preparation to yield an
average protein concentration of
Each assay sample contained 25 µL of supernatant from a cerebral
homogenate, 25 µL (45 pmol) of
[14C]L-arginine, and 100 µL of
reaction buffer (50 mmol/L HEPES, 1 mmol/L EDTA, 1
mmol/L CaCl2, and 1 mmol/L ß-NADPH at pH
7.4). After a 10-minute incubation at 37°C, the reaction was
terminated by addition of 2.0 mL of an ice-cold stop solution
containing 20 mmol/L HEPES and 2 mmol/L EDTA at pH 5.5. The
combined volume was then applied to Poly-Prep
chromatography columns preloaded with 1.0 mL AG 50W-X8
resin (NaOH form; BIO-RAD) and rinsed with 2.0 mL distilled water. This
preparation trapped
Effect of Cofactors on Core and Penumbral NOS Activity
Data Analysis
Core and Penumbral NOS Activity in Control SHR
Core and Penumbral NOS Activity in Isoflurane Sham Groups
Compared with controls, core and penumbral NOS activity was little
affected by administration of 2.5% isoflurane for 1.5 hours (S1.5) or
2 hours (S2.0). However, core and penumbral NOS activity was
significantly increased in the group in which isoflurane was
administered for 1.5 hours and the animals maintained for 24 hours
(S24) compared with the control and other two sham groups
(P<0.05). For example, NOS activity in the S24 group was
higher in each brain region than in the S2.0 group as follows: left
core (76%), left penumbra (26%), right core (18%), and right
penumbra (15%).
Percent Area of Ischemic Injury
Core and Penumbral NOS Activity During MCAO/Reperfusion
Ischemic (Left) Versus Nonischemic (Right)
Hemisphere
Effects of Cofactor Addition on NOS Activity
Isoflurane Sham Groups
To normalize for differences in NOS concentration, we used the NOS
activities observed in the absence of added cofactors and compared them
with the NOS activities present with saturating levels of cofactors
(without/with ratio). This approach eliminated differences between
samples due to differences in NOS concentration and expressed
fractional activation as a function of endogenous cofactor
concentrations. As shown in the top panel of Figure 5
Ischemic Groups
The role of NO as it relates to the evolution of core and penumbral
injury is complex, and an integrated model that takes into account its
neuroprotective and neurotoxic roles has yet to be fully
delineated.1 2 3 4 Protective effects due to
enhancement of endothelial NOS synthesis and NO
production at the vascular level resulting in increased CBF
have been demonstrated.1 2 35 36 Likewise,
neurotoxic effects due to neuronal NOS upregulation and increased
neuronal NO release have also been
shown.1 2 37
Core and Penumbral NOS Activity
Greater NOS activity in the ischemic core may also explain why
the ischemic core expands over time in the absence of
progressively worsening CBF. In the rat filament model of temporary
MCAO, core CBF (lateral caudoputamen and overlying cortex)
during ischemia decreases to approximately 10% of normal and
penumbral flow (adjacent neocortex) to 15% to 20% of
normal.9 Penumbral blood flow may actually
increase between the first (27% of control) and second (36%) hours
after ischemia.11 Other penumbral
regions, including the medial caudoputamen and the
frontoparietal cortex, have even higher CBF values that approach 30%
to 40% of normal.9 10 By most criteria, such
penumbral flow values should not cause infarction unless associated
with some other metabolic
event.17 29 30 31 32 33 34 Also, in models of temporary
MCAO, core and penumbral CBF are rapidly restored early during
recirculation.39 40 Earlier studies have also
shown that NOS-containing neurons are relatively resistant to
hypoxic or ischemic injury and that the neurotoxic effects of
NO are believed to be due to diffusion of NO to adjacent susceptible
neurons.2 3 4 5 Thus, higher core NOS concentrations
could generate an NO gradient particularly near the core-penumbral
interface, where greater amounts of neuronally derived NO could be
released, diffuse to adjacent vulnerable penumbral neurons, and over
time bring about extension of the infarct. Additional evidence
supporting this possibility can be found in recent MCAO studies that
used diffusion-weighted MRI in wild-type versus neuronal NOS–deficient
mice.41 The neuronal NOS–deficient mice had
smaller infarct volumes and smaller peri-infarct (ie, penumbral) zones,
which were believed to be due to reduced NO production.
Several methodological issues need to be considered. The first
concerns the nature of the NOS isoform that we were measuring. It is
likely that we were primarily measuring the neuronal
isoform.8 21 Earlier studies have shown that
inducible NOS is calcium independent and that the time course of
inducible NOS upregulation peaks 2 days after
MCAO.42 In the present study all NOS activity
that we measured was calcium dependent, and we only examined NOS levels
within the first 24 hours after ischemia. It is also unlikely
that endothelial NOS activity was significant because
our studies used only cleared homogenate supernatants that
have been reported to contain little endothelial NOS
activity.43 44
Another issue relates to the extent of regional cerebral differences in
NOS activity and whether there is any rationale for the marked
differences that we observed between core and penumbra. Temporal
profiles of neuronal NOS mRNA after permanent MCAO have shown
upregulation as early as 15 minutes after
occlusion.37 More importantly, the pattern of
increased immunoreactivity corresponded to the ischemic core.
These findings are supported by direct and indirect measurements of NOS
activity. Indirect assays for NO synthesis in rat brain have shown very
high activity in the cerebellum as well as robust striatal
(caudoputamen) and cortical
activity.45 46 Direct regional NOS assays also
have demonstrated greater activity in the cortex and up to 50% higher
striatal NOS activity than other brain
regions.7 47 In addition, other regions within
the rat MCA distribution (hypothalamus, amygdala, and nucleus
accumbens)48 also have greater NOS concentrations
than cortical structures.7 Overall, the available
data suggest that brain regions supplied by the MCA that form the
ischemic core have greater NOS activity than the penumbra.
A related concern is the certainty of our assignment of core and
penumbral regions during dissection. Use of a novel "sandwich"
technique allowed us to measure NOS activity in areas predesignated as
core and penumbra under control, sham, and ischemic conditions
as well as to determine the degree of ischemic injury after
MCAO with the use of TTC staining. In our control and sham groups, MCAO
was not performed, and the dissection of the brain into core and
penumbral regions was based on earlier pharmacological and
histopathologic studies. Primarily on the basis of studies of
pharmacological rescue, it is clear that in the SHR a definable
penumbra exists,16 and as shown in our
TTC-stained prepa-rations, the area of ischemic
mitochondrial injury clearly progressed over the three time periods
included in the present study.
In the ischemic groups, TTC staining was used to substantiate
the presence of ischemic injury before inclusion for core and
penumbral NOS assays.25 26 27 Our measurements of
TTC injury are similar to those reported previously by other
investigators who used both immersion and perfusion
techniques.26 Our data demonstrate a progression
of the area of ischemic injury with time that reflected the
maturation of the ischemic injury into an irreversible infarct.
However, because of the limitations of the TTC (or any) method for
accurate measurement of infarct volume within the first hours after
occlusion, we did not attempt to compare the relation between infarct
volume and core or penumbral NOS activity.
Another issue relates to substrate availability. The marked
pathological demarcation between core and penumbra observed in many
experimental studies has been striking to many
observers.17 18 Because NO synthesis relies on
substrate availability, it is the core-penumbral interface that
theoretically would have relatively higher concentrations of oxygen,
arginine, NADPH, and other necessary cofactors.41
Thus, higher NOS concentrations in the core, coupled with elevated
pericore substrate and cofactor availability, could create a "leading
edge" of NO formation and, depending on local CBF and perfusion
pressure, could have neurotoxic or neuroprotective
effects.49 50 The striking increases in NOS
activity observed with cofactor addition in all regions at all times
support this possibility.
Effects of Cofactor Addition on NOS Activity
Core and Penumbral NOS Activity in Isoflurane Sham Groups
Conclusions
Received August 20, 1997;
revision received February 18, 1998;
accepted February 19, 1998.
2.
Nagafuji T, Sugiyama M, Matsui T, Moto A, Naito S.
Nitric oxide synthase in cerebral ischemia. Mol Chem
Neuropathol. 1995;26:107–157.[Medline]
[Order article via Infotrieve]
3.
Dawson DA. Nitric oxide and focal cerebral
ischemia: multiplicity of actions and diverse outcomes.
Cerebrovasc Brain Metab Rev. 1994;6:299–324.[Medline]
[Order article via Infotrieve]
4.
Iadecola C, Pelligrino, D, Moskowitz MA, Lassen NA.
Nitric oxide synthase inhibition and cerebrovascular regulation.
J Cereb Blood Flow Metab. 1994;14:175–192.[Medline]
[Order article via Infotrieve]
5.
Uemura Y, Kowall NW, Beal MF. Selective sparing of
NADPH-diaphorase-somatostatin-neuropeptide Y neurons in
ischemic gerbil striatum. Ann Neurol. 1990;27:620–625.[Medline]
[Order article via Infotrieve]
6.
Kuppusamy P, Ohnishi ST, Numagami Y, Ohnishi T, Zweier
JL. Three-dimensional imaging of nitric oxide production in the
rat brain subjected to ischemia-hypoxia. J
Cereb Blood Flow Metab. 1995;15:904–913.[Medline]
[Order article via Infotrieve]
7.
Salter M, Duffy C, Garthwaite J, Strubos PJLM.
Substantial regional and hemispheric differences in brain nitric oxide
synthase (NOS) inhibition following
intracerebroventricular administration
of Nw-nitro-L-arginine (L-NA) and its methyl
ester (L-NAME). Neuropharmacology. 1995;34:639–649.[Medline]
[Order article via Infotrieve]
8.
Bredt DS. Molecular characterization of nitric oxide
synthase. In: Vincent SR, ed. Nitric Oxide in the Nervous
System. New York, NY: Academic Press, Inc; 1995:1–42.
9.
Memezawa H, Minamisawa H, Smith ML, Siesjö BK.
Ischemic penumbra in a model of reversible middle cerebral
artery occlusion in the rat. Exp Brain Res. 1992;89:67–78.[Medline]
[Order article via Infotrieve]
10.
Takagi K, Ginsberg MD, Globus MYT, Dietrich D,
Martinez, Kraydieh S, Busto R. Changes in amino acid neurotransmitters
and cerebral blood flow in the ischemic penumbra region
following middle cerebral artery occlusion in the rat: correlation with
histopathology. J Cereb Blood Flow Metab. 1993;13:575–585.[Medline]
[Order article via Infotrieve]
11.
Müller TB, Haraldseth O, Unsgård G.
Characterization of the microcirculation during ischemia and
reperfusion in the penumbra of a rat model of temporary middle cerebral
artery occlusion: a laser Doppler flowmetry study.
Int J Microcirc. 1994;14:289–295.[Medline]
[Order article via Infotrieve]
12.
Brint S, Jacewicz M, Kiessline M, Tanabe J,
Pulsinelli W. Focal brain ischemia in the rat: methods for
reproducible neocortical infarction using tandem occlusion of the
distal middle cerebral and ipsilateral common carotid arteries.
J Cereb Blood Flow Metab. 1988;8:474–485.[Medline]
[Order article via Infotrieve]
13.
Cole DJ, Patel PM, Reynolds L, Drummond JC, Marcantonio
S. Temporary focal cerebral ischemia in spontaneously
hypertensive rats: the effect of ibuprofen on infarct volume.
J Pharmacol Exp Ther. 1993;266:1713–1717.
14.
Morikawa E, Huang Z, Moskowitz MA. L-Arginine decreases
infarct size caused by middle cerebral arterial occlusion
in SHR. Am J Physiol. 1992;263:H1632–H1635.
15.
Zhang F, Iadecola C. Reduction of focal cerebral
ischemic damage by delayed treatment with nitric oxide donors.
J Cereb Blood Flow Metab. 1994;14:574–580.[Medline]
[Order article via Infotrieve]
16.
Ashwal S, Cole DJ, Osborne TN, Pearce WJ. Dual effects
of L-NAME during transient focal cerebral ischemia in the
spontaneously hypertensive rat. Am J Physiol. 1994;267:H276–H284.
17.
Folbergrovà J, Memezawa H, Smith ML, Siesjö
BK. Focal and perifocal changes in tissue energy state during middle
cerebral artery occlusion in normo- and hyperglycemic rats.
J Cereb Blood Flow Metab. 1992; 12:25–33.
18.
Kader A, Frazzini VI, Solomon RA, Trifiletti RR.
Nitric oxide production during focal cerebral ischemia
in rats. Stroke. 1993;24:1709–1716.
19.
Yoshida T, Waeber C, Huang Z, Moskowitz MA. Induction
of nitric oxide synthase activity in rodent brain following middle
cerebral artery occlusion. Neurosci Lett. 1995;194:214–218.[Medline]
[Order article via Infotrieve]
20.
Bredt DS, Snyder SH. Isolation of nitric oxide
synthetase, a calmodulin-requiring enzyme. Proc Natl
Acad Sci U S A. 1990;87:682–685.
21.
Pearce WJ, Tone B, Ashwal S. Maturation alters cerebral
NOS kinetics in the spontaneously hypertensive rat. Am J
Physiol. 1997;42:R1–R7.
22.
Tobin JR, Martin LD, Breslow MJ, Traystman RJ.
Selective anesthetic inhibition of brain nitric oxide synthase.
Anesthesiology. 1994;81:1264–1269.[Medline]
[Order article via Infotrieve]
23.
Ashwal S, Cole DJ, Osborne TN, Osborne S, Pearce WJ.
L-NAME reduces infarct volume in a filament model of transient middle
cerebral artery occlusion in the rat pup. Ped Res. 1995;38:1–5.[Medline]
[Order article via Infotrieve]
24.
Tamura A, Graham DI, McCulloch J, Teasdale GM. Focal
cerebral ischaemia in the rat, I: description of technique and early
neuropathological consequences following middle cerebral artery
occlusion. J Cereb Blood Flow Metab. 1981;1:53–60.[Medline]
[Order article via Infotrieve]
25.
Hatfield RH, Mendelow AD, Perry RH, Alvarez LM, Modha
P. Triphenyltetrazolium chloride (TTC) as a
marker for ischaemic changes in rat brain following permanent middle
cerebral artery occlusion. Neuropathol Appl Neurobiol. 1991;17:61–67.[Medline]
[Order article via Infotrieve]
26.
Bederson J, Pitts L, Germano S, Nishimura M, Davis R,
Bartkowski H. Evaluation of
2,3,5-triphenyltetrazolium chloride as a
stain for detection and quantification of experimental cerebral
infarction in rats. Stroke. 1986;17:1304–1308.
27.
Bartus RT, Dean RI, Cavanaugh K, Eveleth, D, Carriero
DL, Lynch G. Time-related neuronal changes following middle cerebral
artery occlusion: implications for therapeutic intervention and the
role of calpain. J Cereb Blood Flow Metab. 1995;15:969–979.[Medline]
[Order article via Infotrieve]
28.
Swanson RA, Morton MT, Tsao-Wu G, Savalos RA, Davidson
C, Sharp FR. A semiautomated method for measuring brain infarct volume.
J Cereb Blood Flow Metab. 1990;10:290–293.[Medline]
[Order article via Infotrieve]
29.
Hossmann KA. Viability thresholds and the penumbra of
focal ischemia. Ann Neurol. 1994;36:557–565.[Medline]
[Order article via Infotrieve]
30.
Obrenovitch TP. The ischaemic penumbra: twenty years
on. Cerebrovasc Brain Metab Rev. 1995;7:297–323.[Medline]
[Order article via Infotrieve]
31.
Siesjö BK. Pathophysiology and treatment of focal
cerebral ischemia, part 1: pathophysiology. J
Neurosurg.. 1992;77:169–184.[Medline]
[Order article via Infotrieve]
32.
Siesjö BK. Pathophysiology and treatment of
focal cerebral ischemia, part 2: mechanisms of damage and
treatment. J Neurosurg.. 1992;77:337–354.[Medline]
[Order article via Infotrieve]
33.
Back T, Ginsberg MD, Dietrich WD, Watson BD. Induction
of spreading depression in the ischemic hemisphere following
middle cerebral artery occlusion: effect on infarct morphology.
J Cereb Blood Flow Metab. 1996;16:202–213.[Medline]
[Order article via Infotrieve]
34.
Ito U, Kirino T, Kuroiwa T, Klatzo I. Maturation
Phenomenon in Cerebral Ischemia, II. Berlin, Germany:
Springer-Verlag; 1997:225.
35.
Lo EH, Hara H, Rogowska J, Trocha M, Pierce AR,
Huang PL, Fishman MC, Wolf GL, Moskowitz MA. Temporal correlation
mapping analysis of the hemodynamic penumbra in
mutant mice deficient in endothelial nitric oxide
synthase gene expression. Stroke. 1996;27:1381–1385.
36.
Zhang ZG, Chopp M, Zaloga C, Pollock JS, Forstermann U.
Cerebral endothelial nitric oxide synthase expression
after focal cerebral ischemia in rats. Stroke. 1993;24:2016–2021.
37.
Zhang ZG, Chopp M, Gautam S, Zaloga C, Zhang RL,
Schmidt HH, Pollock JS, Forstermann U. Upregulation of neuronal nitric
oxide synthase and mRNA, and selective sparing of nitric oxide
synthase-containing neurons after focal cerebral ischemia in
rat. Brain Res. 1994;654:85–95.[Medline]
[Order article via Infotrieve]
38.
Charriaut Marlangue C, Margaill I, Borrega F, Plotkine
M, Ben Ari Y. NG-Nitro-L-arginine methyl ester
reduces necrotic but not apoptotic cell death induced by
reversible focal ischemia in rat. Eur J
Pharmacol. 1996;310:137–140.[Medline]
[Order article via Infotrieve]
39.
Tsuchidate R, He QP, Smith ML, Siesjo BK. Regional
cerebral blood flow during and after 2 hours of middle cerebral artery
occlusion in the rat. J Cereb Blood Flow Metab. 1997;17:1066–1073.[Medline]
[Order article via Infotrieve]
40.
Liu Y, Mituska S, Hashizume K, Hosaka T, Nukui H. The
sequential change of local cerebral blood flow and local cerebral
glucose metabolism after focal cerebral ischaemia and
reperfusion in rat and the effect of MK-801 on local cerebral glucose
metabolism. Acta Neurochir (Wien). 1997;139:770–779.[Medline]
[Order article via Infotrieve]
41.
Zaharchuk G, Hara H, Huang PL, Fishman MC, Moskowitz
MA, Jenkins BG, Rosen BR. Neuronal nitric oxide synthase mutant mice
show smaller infarcts and attenuated apparent diffusion coefficient
changes in the peri-infarct zone during focal cerebral
ischemia. Magn Reson Med. 1997;37:170–175.[Medline]
[Order article via Infotrieve]
42.
Iadecola C, Xu X, Zhang F, el Fakahany EE, Ross ME.
Marked induction of calcium-independent nitric oxide synthase activity
after focal cerebral ischemia. J Cereb Blood Flow
Metab. 1995;15:52–59.[Medline]
[Order article via Infotrieve]
43.
Sessa WC, Barber CM, Lynch KR. Mutation of
N-myristoylation site converts endothelial
cell nitric oxide synthase from a membrane to a cytosolic protein.
Circ Res. 1993;72:921–924.
44.
Hecker M, Mulsch A, Busse R. Subcellular
localization and characterization of neuronal nitric oxide synthase.
J Neurochem. 1994;62:1524–1529.[Medline]
[Order article via Infotrieve]
45.
Bredt DS, Glatt CE, Hwang PM, Fotuhi M, Dawson TM,
Snyder SH. Nitric oxide synthase protein and mRNA are discretely
localized in neuronal populations of the mammalian CNS together with
NADPH diaphorase. Neuron. 1991;7:615–624.[Medline]
[Order article via Infotrieve]
46.
Förstermann U, Gorsky LD, Pollock JS, Schmidt HH,
Heller M, Murad F. Regional distribution of EDRF/NO-synthesizing
enzyme(s) in rat brain. Biochem Biophys Res Commun. 1990;168:727–732.[Medline]
[Order article via Infotrieve]
47.
Barjavel MJ, Bhargava HN. Nitric oxide synthase
activity in brain regions and spinal cord of mice and rats: kinetic
analysis. Pharmacology. 1995;50:168–174.[Medline]
[Order article via Infotrieve]
48.
Scremin OU. Cerebral vascular system. In: Paxinos G,
ed. The Rat Nervous System. 2nd ed. New York, NY: Academic
Press, Inc; 1995:3–35.
49.
Mulhonen M, Heisted DD, Faraci FM, Loftus CM.
Augmentation of blood flow through cerebral collaterals by inhibition
of nitric oxide synthase. J Cereb Blood Flow Metab. 1994;14:704–714.[Medline]
[Order article via Infotrieve]
50.
Watkins LD. Nitric oxide and cerebral blood flow: an
update. Cerebrovasc Brain Metab Rev. 1995;7:324–337.[Medline]
[Order article via Infotrieve]
51.
Terasako K, Nakamura K, Miyawaki I, Toda H, Kakuyama M,
Mori K. Inhibitory effects of anesthetics on cyclic
guanosine monophosphate (cGMP) accumulation in rat cerebellar slices.
Anesth Analg. 1994;79:921–926.
52.
Rengasamy A, Ravichandran LV, Reikersdorfer CG, Johns
RA. Inhalational anesthetics do not alter nitric oxide synthase
activity. J Pharmacol Exp Ther. 1995;273:599–604.
Department of Internal Medicine,
Cardiovascular Division,
University of Iowa College of Medicine,
Iowa City, Iowa
The role of NO in cerebral ischemia is complex, with the potential for
both protective and detrimental effects.1 2 NO produced by
eNOS appears to play a protective role during and after cerebral
ischemia.1 Potential protective effects include
vasodilatation, inhibition of aggregation of platelets, and inhibition
of expression of redox-sensitive genes such as vascular cell adhesion
molecule-1 (an endothelial-leukocyte adhesion molecule).2
In contrast, nNOS appears to contribute to injury following
ischemia.3 Cytotoxic effects of NO may be mediated by the
reaction of NO with superoxide anion, resulting in the formation of
peroxynitrite, a potent oxidant that can nitrosylate proteins and
damage DNA.1 2
The production of NO and L-citrulline from
L-arginine by NOSs requires the presence of
L-arginine (the substrate), molecular oxygen, NADPH,
tetrahydrobiopterin, FAD, and FMN.4 Interestingly, NOSs
can generate superoxide and hydrogen peroxide in the absence of
adequate levels of L-arginine or
tetrahydrobiopterin.1 5
The present study suggests that cerebral ischemia reduced activity of
NOSs (measured as the conversion of L-arginine to
L-citrulline in brain homogenates in vitro) in both the
ischemic core and penumbra of chronically hypertensive rats.
The addition of enzyme cofactors (tetrahydrobiopterin, FAD, FMN) to the
assay significantly increased activity of NOS in both brain regions,
particularly after 24 hours of reperfusion. Thus, the availability of
enzyme cofactors may be limiting for production of NO. The authors
speculate that higher levels of NOS activity in the ischemic core may
contribute to increased vulnerability of this region and suggest that
regulation of availability of NOS enzyme cofactors may be an
alternative therapy to stroke.
Although the suggestion of regulation of intracellular levels of
NOS cofactors is attractive initially, the effect of manipulation of
these cofactors on brain injury after ischemia is difficult to predict.
If cofactor availability is restricted, nNOS may produce more
superoxide, which could be detrimental. Alternatively, if cofactor
availability is increased, nNOS may produce more NO, which could be
protective under some conditions (by inactivation of superoxide by NO
if subsequent degradation products are nontoxic) but might also be
maladaptive, since nNOS is generally thought to contribute to injury
following ischemia.1 3 In addition, enzyme cofactors could
have additional effects unrelated to NOS. For example, high
concentrations of exogenous tetrahydrobiopterin can produce superoxide
directly through auto-oxidation.6
Received August 20, 1997;
revision received February 18, 1998;
accepted February 19, 1998.
2.
Faraci FM, Heistad DD. Regulation of the cerebral circulation:
role of endothelium and potassium channels. Physiol Rev. 1998;78:53–97.
3.
Huang Z, Huang PL, Panahain N, Dalkara T, Fishman MC, Moskowitz
MA. Effects of cerebral ischemia in mice deficient in neuronal nitric
oxide synthase. Science. 1994;265:1883–1885.
4.
Knowles RG, Moncada S. Nitric oxide synthases in mammals.
Biochem J. 1994;298:249–258.
5.
Xia Y, Dawson VL, Dawson TM, Snyder SH, Zweier JL. Nitric oxide
synthase generates superoxide and nitric oxide in arginine-depleted
cells leading to peroxynitrite-mediated cellular injury.
1995;93:6770–6774.
6.
Tsutsui M, Milstien S, Katusic ZS. Effect of
tetrahydrobiopterin on endothelial function in canine middle cerebral
arteries. Circ Res. 1996;79:336–342.
© 1998 American Heart Association, Inc.
Original Contributions
Core and Penumbral Nitric Oxide Synthase Activity During Cerebral Ischemia and Reperfusion
Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Background and Purpose—The
present studies examined the hypothesis that the distribution of
cerebral injury after a focal ischemic insult is associated
with the regional distribution of nitric oxide synthase (NOS)
activity.
Key Words: cerebral ischemia, focal • cerebrovascular disorders • ischemia • nitric oxide • nitric oxide synthase • rats
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
The neurotoxic
effects of NO during focal cerebral ischemia are well
recognized.1 2 3 4 Inhibition of neuronal NOS or use
of neuronal NOS knockout models during MCAO reduces penumbral infarct
volume with little if any reduction of the infarct core. Regional
differences in NOS histochemical staining with the use of
NADPH-diaphorase have also shown correlation with the
regional vulnerability to ischemic
injury.5 More recently, regional differences in
cerebral NOS activity have also been demonstrated, with greatest
activity in the cortex, striatum, hippocampus, hypothalamus, amygdala,
and substantia nigra.6 7 8 These observations
raise the question of whether there are differences in regional NOS
activity, specifically in core and penumbral regions, and if so whether
this may in part be responsible for the differences in the degree of
ischemic vulnerability and subsequent brain injury. Although
reduced CBF in core regions during ischemia is the major factor
responsible for necrotic injury, other factors, including metabolic rate, capillary density,
excitatory amino acid receptor concentration, and possible local
regional differences in NOS activity, may also contribute to either the
severity or progression of injury. 
) and,
as the duration of ischemia increases, spreads to the overlying
cortex.9 10 The penumbra involves the adjacent
ventrolateral neocortex. Core CBF in this model is reduced to near
constant values at 20, 60, and 120 minutes of occlusion, averaging
approximately 10% of normal with penumbral flow approximately 15% to
20% of normal.9 11 In SHR models of permanent or
temporary MCAO, reduction in infarct volume in the range of 31% to
72% has been reported in response to hypothermia, hemodilution,
L-NAME, L-arginine, 3-morpholino-sydnonimine, ibuprofen,
and tumor necrosis factor-
.12 13 14 15 16 Almost all
recovery has been observed in (and thus pharmacologically defines) the
ischemic penumbra and appears similar in magnitude to that
observed in other rodent species.17
View larger version (43K):
[in a new window]
Figure 1. Depiction of the 4-mm coronal section (section 2)
from which core and penumbral tissues were taken for NOS assays.
Percent area of ischemic injury for section 2 was indirectly
estimated with the use of the TTC area of pallor that reflected
ischemic injury determined from the posterior surface of
section 1 (3 mm) and the anterior surface of section 3 (3
mm).
Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
NOS activity was determined in adult SHR with a modified version
of the method reported by Bredt and Snyder20 in
core and penumbral regions in four separate series of
experiments.21 In the first group (n=12), animals
were anesthetized and immediately decapitated, and NOS activity
was measured from right and left hemisphere brain regions that
corresponded to the core (lateral striatum and overlying cortex) and
penumbra (adjacent ventrolateral cortex), as described in detail below.
The three other groups consisted of SHR undergoing 90 minutes of MCAO
and either no reperfusion (R0), 0.5 hour of reperfusion (R0.5), or 24
hours of reperfusion (R24). Additional sham controls were done for the
total time period of MCAO and reperfusion for these three groups (S1.5,
S2.0, S24), and NOS activity was measured in designated core and
penumbral regions to control for anesthetic effects on NOS activity.
These studies served as controls for the effects of stress and surgery
and also to determine whether inhalation anesthetics such as
isoflurane, which have been shown to alter NOS activity, had similar
effects in the current experimental
protocols.22
Male SHR (weight, 325 to 400 g; n=78) were
anesthetized with 2.5% isoflurane (Forane) and a 60%/40%
air/oxygen mixture. SHR were intubated and mechanically ventilated with
a Harvard rodent respirator at 60 breaths per minute and a tidal volume
of 10 mL/kg body wt, as previously reported.13
End-tidal isoflurane concentration (2.0% to 2.5%) was determined with
a Puritan-Bennett Datex Capnomac infrared anesthetic gas
analyzer. Catheters were inserted into the femoral artery to
monitor mean arterial pressure and for blood sampling and
into the femoral vein for fluid administration. Arterial
blood gas determinations, blood glucose concentration, and hematocrit
were measured throughout the experiments. Pericranial temperature was
maintained at 37°C throughout all experimental procedures.
In each animal the brain was sectioned into three slices
beginning 3 mm from the anterior tip of the frontal lobe (Figure 1
). Section 2 (4 mm thick) was used for measurement of NOS
activity, and sections 1 and 3 (3 mm thick) were used for
estimating the area of mitochondrial ischemic injury. ) to
separate the core (ie, striatum and overlying cortex) from the penumbra
(adjacent cortex). As previously discussed, designation of these core
and penumbral regions was based on pharmacological and
histopathological studies by other investigators that defined the core
to include subcortical structures, primarily the lateral
caudoputamen and overlying cortex, whereas the adjacent
ventrolateral cortex was designated as
penumbra.9 10 11 13 14 15 16 17 We made minor modifications
of these estimates of core and penumbra based on our earlier
investigations in which L-NAME reduced infarct volume in the
SHR.16 In these studies, control infarct volume,
determined at 24 hours with TTC staining, measured 29±1% of the left
hemisphere and comprised striatal and overlying and adjacent cortical
regions, whereas in the L-NAME group, infarct volume was 13±2% and
the ischemic core primarily involved the striatum and some
overlying cortex.
Regions predesignated as "core" and "penumbra" from the
left and right hemispheres were assayed in duplicate for NOS activity
determined as the conversion of
[14C]L-arginine (Dupont) to
[14C]citrulline, modified from the method of
Bredt and Snyder.20 In a previously reported
series of experiments, we had validated our assay technique showing
that L-NAME completely inhibited NOS activity, that NOS activity was
calcium dependent, that homogenate protease activity did
not degrade significant amounts of NOS, that back conversion of
citrulline to L-arginine was minimal, and that the NOS
activity that we measured was primarily the neuronal
isoform.21 
150 µg in each 25-µL aliquot of
supernatant. 99.8% of the remaining
[14C]L-arginine with <7%
citrulline capture. The eluted volume, containing
[14C]citrulline, was measured by liquid
scintillation counting. A separate standard curve was run with each
assay to correct for interassay variations in quench and counting
efficiency. Samples and standards were run in duplicate, and NOS
activity was calculated as picomoles per milligram protein per
minute.
Because cofactor levels are generally not saturating, modest
changes in cofactor availability may influence postischemic
NOS activity. To further evaluate this possibility, we repeated the NOS
assays for all regions after adding cofactors to separate
homogenate samples. Our previous studies using SHR whole
brain homogenates found a 255% increase in NOS activity
with the addition of 10 µmol/L BH4, 3 µmol/L FAD, 3
µmol/L FMN, and 264 µmol/L
calmodulin.21 In the present
series only 10 µmol/L BH4, 3 µmol/L FAD, and 3
µmol/L FMN were added because our previous studies showed a
relatively small effect of calmodulin on NOS activity. For
each cofactor assay, the sample contained 25 µL of supernatant from a
cerebral homogenate, 25 µL (45 pmol) of
[14C]L-arginine, 100 µL of
reaction buffer (50 mmol/L HEPES, 1 mmol/L EDTA, 1
mmol/L CaCl2, 1 mmol/L ß-NADPH, 10
µmol/L BH4, 3 µmol/L FAD, and 3 µmol/L FMN at pH 7.4).
To minimize the contributions of interassay variability to our
measurements, these determinations were run in parallel along with
assays done without cofactor addition with samples from the same
animals.
NOS activity data for all four experimental groups as well as
the additional three sham groups were compared by means of a two-way
ANOVA with region and duration of reperfusion as factors. In a separate
analysis, we also examined the effect of cofactors on NOS
activity for all regions and groups. To normalize for intrinsic
differences in maximal NOS activity, cofactor effects were calculated
as ratios of NOS activity observed in the absence of cofactors relative
to that observed after cofactor addition. Because of the skewed
distribution of these ratios, the data were log transformed, which
resulted in a normal distribution. The resulting transformed ratios
were than subjected to a two-way ANOVA with region and duration of
reperfusion as factors, followed by a Duncan's post hoc
analysis. Unless otherwise indicated, data are expressed as
mean±SEM, and statistical significance was assumed at
P<0.05.
Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
Physiological data (mean±1 SD) for all
experimental groups are summarized in the
Table. Data were obtained during baseline
and occlusion in all groups and at the end of reperfusion in the R0 and
R0.5 groups. For the two 24-hour groups (S24 and R24),
physiological data at the end of reperfusion were
not acquired because these animals no longer had catheters in place.
There were no significant differences in any of the variables when
any of the three sham groups or the three MCAO groups were
compared.
View this table:
[in a new window]
Table 1. Physiological Variables for Sham and MCAO/Reperfusion
Groups
NOS activity was greater in the left (120%) and right (139%)
core regions than in corresponding penumbral regions in the control
group (n=12; P<0.05; Figure 2, top panel). Measurements of NOS
activity (picomoles per milligram per minute) were as follows: left
core, 1392±142; left penumbra, 634±85; right core, 1450±198; and
right penumbra, 606±72. Significant differences were not found when we
compared left versus right core or left versus right penumbra. Combined
(right+left) core NOS (1421±120) was 129% greater than combined
penumbral NOS (620±54) (P<0.05).
View larger version (29K):
[in a new window]
Figure 2. Core and penumbral NOS activity in control (CTL)
(n=12), S1.5 (n=8), S2.0 (n=10), and S24 (n=10) groups. S1.5 SHR were
anesthetized for 1.5 hours, S2.0 SHR were anesthetized
for 120 minutes, and S24 were anesthetized for 1.5 hours and
then recovered for 24 hours. In all groups hemisphere core NOS activity
was greater than the corresponding NOS activity determined from the
penumbra (*P<0.05). Hemispheric differences for core or
penumbral NOS activity were not observed. Addition of cofactors
increased NOS activity in all regions (P<0.05). RCORE
indicates right core; LCORE, left core; RPEN, right penumbra; and LPEN,
left penumbra.
Right and left hemisphere core NOS activity in each of the three
sham groups was greater than the corresponding penumbral NOS activity
(P<0.05; Figure 2, top panel). Differences were not
observed when we compared left versus right core or left versus right
penumbra in any of the three sham groups. Combined core activity in
each of the three sham groups was significantly higher than combined
penumbral activity: S1.5 (208%; n=8), S2.0 (122%; n=10), and S24
(161%; n=10;) (P<0.05). Thus, as in the control groups,
there were no side-to-side differences in NOS activity for either the
core or penumbral regions in any of the sham groups, and NOS activity
was consistently greater in the core than in the penumbral
regions.
The percent area of ischemic injury measured 24±2% after
1.5 hours of occlusion (R0 group; n=13), increased to 28±3% after 0.5
hour of reperfusion (R0.5 group; n=12) and by 24 hours increased to
40±2% (R24 group; n=13; P<0.05 compared with the other
two groups). At the end of occlusion (Figure 3), this area of TTC pallor was located
in the lateral caudoputamen and cortex and by 0.5 hour of
reperfusion had extended more cortically and was more easily
differentiated from nonischemic brain. By 24 hours the
ischemic hemisphere was edematous, and the area of injury had
continued to extend to adjacent regions, as shown.
View larger version (41K):
[in a new window]
Figure 3. Representative TTC-stained brain
sections (section 3) from the three ischemia/reperfusion groups
demonstrating areas of ischemic injury observed at that time:
(A) 1.5 hours of occlusion; (B) 1.5 hours of occlusion and 0.5 hour of
reperfusion; and (C) 1.5 hours of occlusion and 24 hours of
reperfusion. Percent areas of infarction for the three groups averaged
24±2%, 28±3%, and 40±2%, respectively.
Core Versus Penumbral NOS
Both ischemic (left) and nonischemic (right) core
NOS activity remained higher than corresponding penumbral regions at
all times (Figure 4, top panel;
P<0.05). These differences, expressed as the percentage
that core NOS activity was greater than penumbra
[(Core-Penumbra)/Penumbra · 100], averaged 138%, 82%, and
156% in the ischemic hemisphere and 131%, 203%, and 92% in
the nonischemic hemisphere at the end of occlusion (R0; n=13),
30 minutes of reperfusion (R0.5; n=12), and at 24 hours (R24; n=13),
respectively. As shown in Figure 4, even though the overall trend was
for NOS activity to decrease in the ischemic hemisphere and to
increase in the nonischemic hemisphere, the relation between
core and penumbral NOS activity remained relatively constant.
View larger version (30K):
[in a new window]
Figure 4. Core and penumbral NOS activity in control (n=12),
R0 (n=13), R0.5 (n=12), and R24 (n=13) groups (see "Materials and
Methods"). Values for both ischemic (left [L-PEN]) and
nonischemic (right [R-PEN]) penumbral NOS activity were lower
than those for corresponding core regions at all times
(*P<0.05). NOS activity was lower in the
ischemic core (left [L-CORE]) than in the nonischemic
core (right [R-CORE]) in all three MCAO groups
(§P<0.05). NOS activity in the ischemic core
increased 18% from control (CTL) by 1.5 hours of MCAO and then
decreased significantly by 0.5 hour (31%) and 24 hours (78%) of
reperfusion (P<0.05). Corresponding NOS activity in the
nonischemic core increased 60%, 27%, and 51% at these times
compared with controls. Left penumbral and right penumbral NOS activity
decreased by 0.5 hour of reperfusion, but by 24 hours left penumbral
NOS continued to decrease, whereas right penumbral NOS increased above
control values (+). Addition of cofactors increased NOS activity in all
regions (P<0.05) and at all times after
ischemia (P<0.05).
Core and penumbral NOS activity was consistently lower in
the ischemic hemisphere than the nonischemic hemisphere
at all times (Figure 4, top panel; P<0.05). In core
regions, NOS activity was 29%, 48%, and 86% lower in the
ischemic than the nonischemic hemisphere in the R0,
R0.5, and R24 groups. Similar differences in penumbral NOS activity for
the same groups were also observed, ie, 31%, 13%, and 90% lower in
the ischemic versus nonischemic hemisphere.
Controls
Addition of cofactors to separate homogenate samples
from the control group increased NOS activity in all brain regions
(Figure 2, bottom panel). The percent increase in NOS activity between
the "without" cofactors and "with" cofactors groups were as
follows: left core, 258%; left penumbra, 210%; right core, 285%; and
right penumbra, 229% (P<0.05). For all regions combined,
the average was 256%. There were no significant differences in the
percent increase in NOS activity between core and penumbra or between
ipsilateral and contralateral homologous regions. NOS activity after
cofactor addition remained significantly higher in core than in
penumbral regions in both hemispheres (P<0.05).
Addition of cofactors increased NOS activity in all regions in all
sham groups (P<0.05; Figure 2, bottom panel).
Addition of cofactors also had no effect on the right-left differences
in any sham groups. Cofactor addition also appeared to have a greater
effect on total NOS activity in sham-treated animals than controls. For
all brain regions combined, the percent increases in NOS activity in
the three sham groups were 545% (S1.5), 350% (S2.0), and 667% (S24)
compared with the overall increase seen in the control group of 256%
(P<0.05). Even with the addition of cofactors, NOS activity
remained significantly higher in core than in penumbral regions in both
hemispheres (P<0.05). Combined core NOS activity in the
S1.5, S2.0, and S24 groups was 134%, 141%, and 119% higher than the
corresponding combined penumbral NOS activity (P<0.05). , the fractional activation of NOS
supported by endogenous levels of cofactors did not vary
significantly with region or side at any time in the sham groups. This
finding suggests that the increase in total activity observed at 24
hours in the sham animals relative to controls (Figure 2, bottom panel)
cannot be attributed to differences in cofactor availability and must
instead be due to differences in NOS concentration and/or enzyme
specific activity induced by isoflurane.
View larger version (55K):
[in a new window]
Figure 5. Effects of cofactors on NOS activity. Data are
presented as the log transformation of the ratios of NOS
activity observed in the absence of cofactors relative to that observed
after cofactor addition. The top panel shows the ratio data by region
for the control (CTL) and sham groups (see "Materials and Methods")
and reflects the fractional activation of NOS supported by
endogenous levels of cofactors. No significant differences
were observed when we compared the core versus penumbral regions or
left versus right hemispheres. The bottom panel (control and MCAO
groups) shows no differences at the end of occlusion (R0) or early
during reperfusion (R0.5). However, after 24 hours of reperfusion,
values of fractional activation of NOS were dramatically depressed in
both the core and penumbral regions of the infarcted left side compared
with similar regions from the right hemisphere (*) or with similar
regions from the left hemisphere of the sham controls at 24 hours (+).
Abbreviations are as in Figure 4 .
In all groups and at all time points (Figure 4, bottom panel),
addition of cofactors increased NOS activity significantly
(P<0.05). For the four brain regions combined, the increase
in NOS activity in the three MCAO groups averaged 434% (R0), 431%
(R0.5), and 695% (R24). The effect of cofactor addition in these
groups was significantly greater than the overall increase seen in the
control group (256%) (P<0.05). However, as shown in the
bottom panel of Figure 5, the fractional activation of NOS supported by
endogenous levels of cofactors did not vary significantly
with region or side after 0 or 30 minutes of reperfusion but did vary
significantly after 24 hours of reperfusion. In particular, after 24
hours of reperfusion values for fractional activation of NOS were not
significantly different than control values on the noninfarcted right
side but were dramatically depressed in both the core and penumbral
regions of the infarcted left side. These findings indicate that
differences in cofactor availability probably do not contribute to
differences in total NOS activity at the end of occlusion or early
during reperfusion but may be particularly important in infarcted
regions after 24 hours of reperfusion.
Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
The concept of the ischemic penumbra was originally
introduced to define brain regions in which blood flow was insufficient
to sustain cellular electrical activity but above that required to
maintain ionic gradients.29 30 More recently, the
concept of the penumbra was expanded to include those perifocal areas
that are marginally perfused but that may be recruited into the
infarctive process unless perfusion is restored or pharmacological
measures are instituted that prevent additional cell
death.30 Penumbral tissues are considered
metabolically dysfunctional, with survival dependent on
events related to abnormal calcium metabolism, excitotoxic
injury, enhanced free radical production, or spreading
depression that progressively increases cell
injury.30 31 32 33 As yet, events leading to infarct
maturation remain incompletely understood.34
The principal finding of the present study, that NOS activity
is greater in anatomically defined core than in penumbral regions, may
explain in part why the ischemic core is more vulnerable to
ischemic injury than the penumbra. Core NOS activity was 129%
higher in controls and 208%, 122%, and 161% higher in the three sham
groups. The higher NOS activity observed in core compared with
penumbral regions was thus not due to ischemic effects because
it was consistently observed in the control and sham groups.
Likewise, core NOS activity during ischemia and at various
stages of reperfusion remained higher than penumbra in both the
ischemic and nonischemic hemispheres. While it is clear
that the principal reason for core necrotic injury during MCAO relates
to the severity and duration of reduced CBF, other factors appear to
contribute to the cascade of injury that occurs during ischemia
and reperfusion.29 30 Our earlier studies in a
rat model of transient MCAO suggested that the principal neurotoxic
effects of NO occur early during reperfusion, whereas during
ischemia vascular NO might act to ameliorate ischemic
injury by maintaining CBF.16 Thus, the
observations of the present study make the important suggestion
that higher core NOS activity may be an additional critical factor
early during reperfusion that accounts for the preferential
vulnerability of "core" regions compared with "penumbral"
regions. Recent data in a rat model of MCAO, showing that L-NAME, an
inhibitor of NOS, reduced necrotic cell injury in the
ischemic core but not apoptotic cell death in the
penumbra, support these observations.38 However,
it is also important to recognize that the present study examined
NOS activity ex vivo and that this only defines the potential for NO
production and is not necessarily a reliable index of actual NO
output. Additional studies either directly measuring core and penumbral
NO production during ischemia in vivo (eg,
microdialysis) or using cultured cell or brain slice techniques in
conjunction with NOS immunocytochemical studies to detemine the number
of NOS neurons, such as in the study of Zhang et
al,37 would be of importance.
Not unexpectedly, our data demonstrate that cofactor addition
increased NOS activity in all groups. The preservation of the hierarchy
of core over penumbra, even after cofactor addition, lends additional
support to the notion that intrinsic differences in the concentration
of the NOS enzyme or its specific activity are present in these two
regions. To control for the effects of differences in NOS concentration
or specific activity, we normalized NOS activity values relative to the
maximum values observed in the presence of saturating concentrations of
cofactors and thus quantified the fractional activation of NOS
supported by endogenous cofactor concentrations. This
analysis revealed that the fractional activation of NOS
supported by endogenous cofactor concentrations is
relatively constant at all time points and regions in sham animals, as
well as at the end of occlusion and early during reperfusion in
ischemic animals. Thus, at these times differences in total NOS
activity can be attributed only to differences in NOS concentration or
enzyme specific activity. In contrast, after 24 hours of reperfusion,
the fractional activation of NOS supported by endogenous
cofactor concentrations was dramatically depressed, indicating that
cofactor availability is a major determinant of NOS activity in
postischemic infarct regions. Additional studies, measuring
FAD, FMN, and particularly BH4, which is uniquely necessary for NOS,
are needed to clarify exactly which of these cofactors are most limited
in availability.3 4 8
Earlier in vitro investigations have shown either an
inhibitory effect (up to 85%) or no effect of isoflurane
and other volatile anesthetics on brain NOS
activity.51 52 However, in the studies that used
rat brain homogenates, isoflurane was added to the
incubation mixture rather than given in vivo to the intact
animal.52 In the present study (Figure 2, top
panel) we did not find evidence of inhibition of NOS using a sham
protocol when isoflurane was given for 1.5 or 2.0 hours. Surprisingly,
after cofactor addition, significant increases in NOS activity were
observed in the 1.5-hour and 24-hour sham groups. These findings, as
well as others in the literature, suggest that certain anesthetics
might augment as well as inhibit NOS synthesis under different
experimental conditions and point out the need to include appropriate
control groups.
The observation that NOS activity is greater in core regions than
in penumbra in control, sham, and ischemia/reperfusion groups
suggests an additional explanation for the vulnerability of the core to
ischemic and reperfusion injury. Higher core NOS activity may
also contribute to infarct maturation. In addition, the effects of
cofactors on restoring NOS activity in both core and penumbral regions
suggest that regulation of cofactors could provide an alternative
approach to the treatment of stroke.
Selected Abbreviations and Acronyms
BH4
=
tetrahydrobiopterin
CBF
=
cerebral blood flow
FAD
=
flavin adenine dinucleotide
FMN
=
flavin mononucleotide
L-NAME
=
NG-nitro-L-arginine methyl ester
MCA
=
middle cerebral artery
MCAO
=
middle cerebral artery occlusion
NO
=
nitric oxide
NOS
=
nitric oxide synthase
SHR
=
spontaneously hypertensive rats
TTC
=
2,3,5-triphenyltetrazolium chloride
Acknowledgments
This study was supported by a grant from the Pediatric Research
Fund, with additional generous support from the Department of
Anesthesiolgy and the Division of Perinatal Biology at Loma Linda
University School of Medicine.
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
1.
Dalkara T, Moskowitz MA. Neurotoxic and
neuroprotective roles of nitric oxide in cerebral ischemia.
Int Rev Neurobiol. 1997;40:319–336.[Medline]
[Order article via Infotrieve]
Editorial Comment
Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
The brain produces relatively large amounts of nitric oxide under
normal conditions. The two sources of this constitutively produced NO
are endothelial and neuronal isoforms of NO synthase (eNOS and nNOS,
respectively).
Selected Abbreviations and Acronyms
BH4
=
tetrahydrobiopterin
CBF
=
cerebral blood flow
FAD
=
flavin adenine dinucleotide
FMN
=
flavin mononucleotide
L-NAME
=
NG-nitro-L-arginine methyl ester
MCA
=
middle cerebral artery
MCAO
=
middle cerebral artery occlusion
NO
=
nitric oxide
NOS
=
nitric oxide synthase
SHR
=
spontaneously hypertensive rats
TTC
=
2,3,5-triphenyltetrazolium chloride
References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Introduction
References
1.
Iadecola C. Bright and dark sides of nitric oxide in
ischemic brain injury. Trends Neurosci. 1997;20:132–139.[Medline]
[Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  H. Hong, J.-S. Zeng, D. L. Kreulen, D. I. Kaufman, and A. F. Chen Atorvastatin protects against cerebral infarction via inhibition of NADPH oxidase-derived superoxide in ischemic stroke Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2210 - H2215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Fitzpatrick, Y. Shi, W. C. Hutchins, J. Su, G. J. Gross, B. Ostadal, J. S. Tweddell, and J. E. Baker Cardioprotection in chronically hypoxic rabbits persists on exposure to normoxia: role of NOS and KATP channels Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H62 - H68. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Shi, W. C. Hutchins, J. Su, D. Siker, N. Hogg, K. A. Pritchard Jr., A. Keszler, J. S. Tweddell, and J. E. Baker Delayed cardioprotection with isoflurane: role of reactive oxygen and nitrogen Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H175 - H184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Veltkamp, N. Rajapakse, G. Robins, M. Puskar, K. Shimizu, and D. Busija Transient Focal Ischemia Increases Endothelial Nitric Oxide Synthase in Cerebral Blood Vessels Stroke, November 1, 2002; 33(11): 2704 - 2710. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. S. Bonser, C. H. Wong, D. Harrington, D. Pagano, M. Wilkes, T. Clutton-Brock, and M. Faroqui Failure of retrograde cerebral perfusion to attenuate metabolic changes associated with hypothermic circulatory arrest J. Thorac. Cardiovasc. Surg., May 1, 2002; 123(5): 943 - 950. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Castillo, R. Rama, and A. Davalos Nitric Oxide-Related Brain Damage in Acute Ischemic Stroke Stroke, April 1, 2000; 31(4): 852 - 857. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gbadegesin, S. Vicini, S. J. Hewett, D. A. Wink, M. Espey, R. M. Pluta, and C. A. Colton Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death Am J Physiol Cell Physiol, October 1, 1999; 277(4): C673 - C683. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||