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Diffuse cerebral ischemia in the cat II. Regional metabolites during severe ischemia and recirculation

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Diffuse Cerebral Ischemia in the Cat:
II. Regional Metabolites During Severe
Ischemia and Recirculation
Frank A. Welsh, PhD, Myron D. Ginsberg, MD, Wendy Rieder, BS,
and William W. Budd, BS
Metabolite levels were measured in seven brain regions in cats after 15 or 30 minutes of a severe ischemic insult and
after a 90-minute period of recirculation following 15 or 30 minutes of ischemia. Brain levels of phosphocreatine were
depleted after a 15- or 30-minute insult, and lactate levels were extremely high at both times. The adenosine
triphosphate (ATP) content in many brain areas and the presence of microregions of low reduced nicotinamineadenine dinucleotide in the brains of the animals that had 15 minutes of ischemia suggested that the ischemia, though
severe, was not complete.
Recirculation following a 15-minute insult restored brain levels of ATP and phosphocreatine to 70 to 100% of
control values in all regions analyzed. In contrast, metabolic recovery from a 30-minute insult was regionally
heterogeneous. Thus, there was persistent depression of ATP and phosphocreatine and elevation of lactate, which
was localized in discrete cortical foci near the longitudinal midline. The factors governing the localization of
metabolic failure must have become manifest during the recirculation period since the ischemic insult itself caused
similar metabolic perturbations in all cortical regions.
Welsh FA, Ginsberg MD, Rieder W, et al: Diffuse cerebral ischemiain the cat: 11. Regional metabolites during
severe ischemia and recirculation. Ann Neurol 3:493-501, 1978
The ability of the brain to recover from a period of
ischemia is limited compared with other organs, and
the factors restricting recovery are still not well understood. Of the many mechanisms that might become damaged during a period of ischemia, those
involved in energy metabolism are perhaps the most
fundamental to viability of the tissue. Furthermore,
adequate delivery of glucose and oxygen following an
ischemic episode is a prerequisite for restoration of
energy metabolism. In the preceding paper (p 482,
this issue), we report regionally nonhomogeneous
reperfusion following a 30-minute ischemic insult,
which contrasted with homogeneous though diminished reperfusion when the insult was limited t o
1 5 minutes. In this study, we examined the recovery
of metabolite levels in several regions of brains subjected to 15 or 30 minutes of ischemia in order to
determine the metabolic correlates of these perfusion states.
Sixteen pentobarbital-anesthetized cats were subjected
to periods of severe cerebral ischemia, followed in some
cases by a 90-minute period of normotensive recirculation.
Five animals received 15-minute ischemic insults and 5,
30-minute insults, all without subsequent recirculation.
Three animals received 15 minutes of ischemia followed by
recirculation, and 3, 30 minutes of ischemia followed by
recirculation. Three control animals were studied after a
comparable period of anesthesia but without ischemia. The
procedures for animal preparation are described in the preceding report [3]. Table 1 summarizes the physiological
variables (blood pressure, arterial blood gas tension, and
pH) for the animals in the present study.
Cerebral ischemia was produced by occlusion of the
common carotid arteries and the basilar artery, followed by
rapid (1- to 3-minute) arterial hemorrhage into heparinized
syringes. Mean arterial pressure was reduced until an
isoelectric EEG appeared (mean arterial pressure, 70 to 80
mm Hg) and was controlled at that level by withdrawing or
reinfusing small quantities of blood through a femoral artery catheter. I n animals undergoing postischemic recirculation, the vascular clips were removed and the shed blood
reinfused over 5 to 7 minutes to restore normotension (see
Table 1). At the conclusion of each experiment, the cats
were inverted from supine to prone position, and the brain
was frozen in situ for 10 to 15 minutes by pouring liquid
nitrogen into a Styrofoam cup fixed to the exposed calvarium. During the period of freezing, animals were ventilated mechanically and normal blood pressures were main-
From the Division of Neurosurgery and the Cerebrovascular Research Center of the Department of Neurology, UniverSiV of
Pennsylvania School of Medicine, Philadelphia, PA.
Address reprint requests to Dr Ginsberg, Department of Neurology, Hospital of the University of Pennsylvania, 3400 Spruce St.
Philadelphia, PA 19104.
Methods
Accepted for publication Jan 3, 1978.
0364-5134/78/0003-0605$01.50@ 1978 by Frank A. Welsh
493
Table I . Physiological Data”
No Recovery
Determination
Preinsult period
MABP
Po,
Pco*
PH
Control
Grow
15 Min
Ischemia
30 Min
Ischemia
15 Min
Ischemia
30 Min
Ischemia
146 f 16
165 2 5
32.6 f 1.3
7.341 t 0.030
134 f 14
125 f 8
32.7 ? 0.8
7.345 f 0.012
130 f 13
139 2 9
32.0 f 0.5
7.308 ? 0.017
195 f 10
137 t 7
33.4 2 0.7
7.315 2 0.017
145 ? 13
139 t 5
31.4 ? 1.6
7.329 f 0.006
Insult
MABP
...
Postinsult period
MABP
Po,
Pco2
PH
...
...
...
...
‘Mean values are given as mm Hg
68
?
72 f 3
2
...
...
...
...
%
...
...
...
...
78
f7
157 t 13
132 ? 5
32.5 f 0.6
7.343 t 0.008
75 2 4
148 2 7
133 f 5
32.7 f 2.7
7.317 f 0.028
standard error.
MABP = mean arterial blood pressure (diastolic pressure
+ pulse pressure/3).
tained. This freezing technique traps metabolites in deep
structures of cat brain without ischemic alteration [16- 181
(see Tables 2, 3). Thus, deep brain regions continue to be
perfused until the time of tissue freezing.
The methods for brain freezing, sectioning, lowtemperature reduced nicotinamide-adenine dinucleotide
( NADH) fluorescence photography, regional sampling,
and determination of metabolite levels have been described previously in detail [ 15, 181. Briefly, the brain, frozen in liquid nitrogen, was sectioned in the coronal plane at
- 196°C. Frozen slices were illuminated with 366 nm excitation light, and the 450 nm fluorescence signal, which is
derived primarily from N A D H , was recorded photographically. At -30°C the slices were sampled regionally
(Fig l ) , weighed, and extracted in alkaline methanol. The
extract was divided and one portion was heated prior to
analysis for N A D H content [5]. The other portion was
acidified and centrifuged, and the neutralized supernatant
was analyzed for adenosine triphosphate (ATP), phosphocreatine, and lactate [7]. Data were analyzed for statistical
significance by a one-way analysis of variance.
Results
Metabolic Alterations during Cerebral Ischemia
Table 2 compares the regional metabolite levels after
15 or 30 minutes of ischemia with control values.
Phosphocreatine levels were nearly depleted in all
regions analyzed after 15 or 30 minutes of ischemia.
Lactate levels approached 30 mmol per kilogram of
brain sample weight in gray matter regions at both
times, while in white matter, lactate levels did not
exceed 20 mmol per kilogram. Likewise, N A D H
levels during ischemia increased to higher levels in
gray than in white matter. ATP levels in the two
white matter regions fell to 1.2 mmol per kilogram at
15 minutes and had not declined further by 30 min-
494
Recovery
Annals of Neurology Vol 3
No 6 June 1978
F i g 1 . Location and size of regional samples analyzedfor
metabolite content: M = medialgyrus; L = lateralgyrus; S =
J-ulcus;W = subcortical white matter; C = cingukzte gyrus;
B = basalganglia (caudate nucleus); I = internal capsule. The
rmprint of the sampling punch marks the seven regions sampled
bikz terally .
utes. In gray matter there was considerable variation
in ATP levels among individual animals as well as
from one region to another in the same animal. Figure 2 displays the ATP level of each cortical sample
after 15 or 30 minutes of ischemia in the present
model; cortical ATP levels resulting from complete
ischemia produced by cardiac arrest are presented for
comparison. At 1 5 minutes the level of ATP in 26 of
the 40 cortical samples was greater than 0.5 mmol per
kilogram. Since 5 minutes of complete ischemia was
sufficient to reduce ATP levels below 0.5 mmovkg, it
Table 2 . Effect of 15
OY
30 Minutes of Ischemia on Regional Metabolite Levelsa
Region
Metabolite
Control
15-Min Insult
30-Min Insult
Lateral gyms
ATP
PCr
Lac
NADH
2.29 f 0.02
5.40 f 0.27
0.63 ? 0.14
10.5 f 1.7
0.76 f 0.17
0.13 ? 0.03
30.2 f 2.2
42.6 ? 13.6
0.26 -c 0.12
0.09 r 0.04
32.2 f 2.2
48.1 ? 12.3
Medial gyms
ATP
PCr
Lac
NADH
2.33 ? 0.06
5.65 c 0.14
0.69 f 0.15
8.7 1.3
*
1.03 f 0.31
0.46 ? 0.26
29.0 f 2.7
31.8 f 8.8
0.46 0.26
0.49 t 0.39
32.3 c 4.0
38.4 -c 12.0
sulcus
ATP
PCr
Lac
NADH
2.21 f 0.05
4.92 t 0.25
1.04 2 0.13
16.2 & 3.0
0.76 f 0.23
0.33 f 0.15
30.5 f 2.7
42.3 f 7.1
0.46 f 0.21
0.36 f 0.24
31.2 f 3.2
48.2 & 10.0
Cingulate gyms
ATP
PCr
NADH
2.44
5.58
0.82
7.2
0.26
? 0.14
f 0.4
0.76 ? 0.33
0.16 f 0.13
31.1 f 3.8
44.9 f 9.6
0.13 ? 0.04
0.09 f 0.08
33.6 f 4.2
45.4 c 12.1
White matter
ATP
PCr
Lac
NADH
2.06 f 0.04
3.11 ? 0.07
0.70 f 0.17
7.9 2 0.6
1.17 f 0.07
0.06 ? 0.01
16.6 f 1.2
16.3 f 1.9
1.06 f 0.17
0.20 f 0.12
19.8 ? 2.7
13.1 f 2.9
Internal capsule
ATP
PCr
1.95 & 0.05
3.45 ? 0.35
0.48 ? 0.07
4.6 f 0.2
1.14 f 0.05
0.15 f 0.05
15.8 2 1.4
14.9 ? 1.5
1.31 ? 0.22
0.57 ? 0.32
15.6 & 2.6
11.4 2 1.7
2.48 f 0.06
6.30 c 0.40
0.86 & 0.15
12.5 t 2.0
0.74 f 0.26
0.13 ? 0.08
30.5 f 2.7
55.6 ? 15.0
0.27 2 0.10
0.11 f 0.05
33.3 f 3.9
60.4 f 16.1
Lac
Lac
NADH
Caudate nucleus
ATP
PCr
Lac
NADH
'Values are mean
analyzed.
ATP
?
f 0.05
?
standard error. ATF', PCr, and Lac are given in mmdkg, NADH in pmoVkg. See Figure 1 for location of regions
= adenosine triphosphate; PCr = phosphocreatine; Lac = lactate;
is evident that the present model of ischemia did not
completely abolish flow by 15 minutes. However, by
30 minutes there were only a few cortical regions
with ATP values greater than 0.5 mrnollkg.
Figure 3 depicts the effects of ischemia o n brain
NADH fluorescence, In control brains such as the
one shown in Figure 3A, low levels of fluorescence
were present in all gray matter regions except for the
depths of several cortical sulci (region S, Fig 1).
These sulci are the only regions of control brain that
suffer ischemic metabolic perturbation due to the
freezing process [ l b , 181. Thus, the levels of NADH
and lactate were higher and the level of phosphocreatine was lower in the sulcus than in other regions
of gray matter (see Table 2).
After 15 minutes of ischemia there was a patchy
increase of NADH (Fig 3B) in the cerebral cortex
and caudate nucleus in 4 of the 5 animals. In the
NADH
=
reduced nicotinamide-adenine dinucleotide.
remaining animal of the 15-minute ischemia group,
NADH was diffusely elevated throughout the gray
matter structures. Figure 4 shows the regional levels
of ATP and NADH in the brain slice pictured in
Figure 3B. ATP values ranged from 0.3 to 1.5
mrnoVkg in cortex and measured 0.4 mmoykg in
caudate and 1.2 mmoVkg in white matter. Gray matter NADH levels, measured enzymatically, correlated well with the intensity of NADH fluorescence
(see Fig 3B) and varied inversely with the tissue content of ATP. White matter, which has high background fluorescence 1181, contained lower amounts
of NADH than did gray matter, although the levels
were two to three times those in control white matter
(see Table 2).
After 30 minutes of ischemia there was a diffuse
increase of NADH fluorescence (Fig 3C) in 4 of the
5 animals, with the remaining cat showing a patchy
Welsh et al: Metabolites in Diffuse Brain Ischemia 495
S
10
IS
20
30
YINUTLS OF ISCNEYIA
Fig 2. Regionallevels of ATP in cortex during cmbralischemia.
The dosed rirdcr repmetat ATP heh in cwtiull s a m p h a h
15 minntes (5 animah) w 30 minntes (5 animah) of iScbemia
a pnulucedin the present modrlof vascular occlusion andsystemic bypotension.For comparison,m p k k iscbemia w a pro~
dncad b~ canliar a m : in 4 animak After 5 minntes of a m
flow, the brains of 3 of tbe animah wnrf;ozm and eigbt c o d calsampks per animal (INPig I ) were anat'yzedjirATP content. Tbe brain of tbefourtb zetv-flow animalwasfi.oem a$&
30 minutes. Tbe dottedama between the lines marked cardiac
arrest represents 2 I SD fiom the mean. LiktwiSe, the control
zone reptvsents 2 I SD fiorrr the mean. Valnesam in m d l k g .
pattern of fluorescence. Figure 5 shows the levels of
ATP and NADH in the brain slice pictured in Figure
3C. ATP levels were nearly depleted in all gray matter regions but were only 50% reduced from control
levels in white matter. NADH levels were increased
tenfold in cortex and caudate compared to a two to
rhreefold increase in'white matter. Thus, the pattern
ofdiffrrsc
gray matter fluorescence (Fig 3C) characteristic of a 30-minute insult correlated with lower ATP
levels and higher NADH levels than the patchy increase of NADH (Fig 3B, 4) that typified the 15minute ischemic insult.
Metabolic Recovery from Cmbral Ischemia
In animals that had 90 minutes of recirculation following a 15-minute ischemic insult, metabolite levels
returned toward control values in all regions studied
(Table 3). Mean ATP levels ranged from 67 to 74%
of control in gray matter and were 88% ofcontrol in
white matter. Phosphocreatine values recovered to
70 to 80% of control in gray matter and 90 to 100%
in white matter. Gray matter lactate and NADH
averaged six and two times control values, respectively, while the white matter content of these two
metabolites returned to control levels. Thus,metabolite values 90 minutes after a 15-minntr ischemic insult recovered greatly but did not return to normal,
especially in gray matter regions.
496 Annals of Neurology Vol 3 No 6 June 1978
L
F i g 3. Representativephotagraphsof NADH fluomtcenn(4SO
nm) i n fmzen sections of rat brain. (A)Controlbrain.(B)
Patchy flnomtcence pattem in a brain ischemicfor I5 minntes.
(C) Dgfaselyflrrorescentgray matter regions in a brain s n b
jectedto 30 minntes of ischemia.
ATP
...
.
NADH
83w78
-
Fig 4 . Regional levels ofATP and NADH after 15 minutes of
ischemia. The brain pictured in Figure 3 8 was sampled regionally. ATP vdues am in mmollkg, NADH values in pmoll
kg. Each value represents the mean of duplicate assays on a
single sample.
Fig 5 . Regionallwels ofATPandNADH after30 minutesof
ischemia. The brain pictured in Figure 3C was sampled regionalb. ATP values are i n mmollkg, NADH values in pmoll
kK. Each value represents the mean of duplicate assays on a
single rample.
With recirculation following a 30-minute ischemic
insult, however, recovery of metabolite levels in the
sulcus and medial gyms (regions S and M in Fig 1)
was slight compared with the other brain regions
analyzed (see Table 3). Figure 6 compares the recovery of ATP levels in four cortical regions following
15 or 30 minutes of cerebral ischemia and 90 minutes
of recirculation. ATP levels in the sulcus and medial
gyms were significantly (p < 0.05) lower than those
of neighboring cortical areas (lateral and cingulate
gyri) during recovery from 30 minutes of ischemia.
However, when the ischemic insult was limited to 15
minutes, ATP levels following recirculation returned
to 60 to 70% of control levels in all four cortical
regions.
I n addition to the regional heterogeneity of metabolic recovery after a 30-minute insult, heterogeneity
of blood content was evidenced by visual inspection
of the frozen brain slices. Figure 7 shows that the
cerebral cortex contained blanched regions that were
frequently rimmed by hyperemia. The regions with
the greatest degree of blanching, the medial gyri and
adjacent sulci, contained the lowest levels of ATP
and phosphocreatine and the highest levels of tissue
lactate (Fig 8). However, N A D H content as well as
tissue fluorescence were below normal in the
blanched cortex. In the lateral gyri, which were
hyperemic, ATP and phosphocreatine had returned
to 50% of control levels while lactate remained extremely elevated. Metabolic recovery was more advanced in the white matter and caudate nucleus than
in any region of cerebral cortex.
Discussion
In the present model, cerebral ischemia of 15 or 30
minutes’ duration produced profound alterations of
high-energy phosphates, lactate, and N A D H in all
brain regions analyzed. In the accompanying paper
[ 3 ] , mean blood flow measured with the 14Cantipyrine autoradiographic technique was less than 1
to 2% of control flow values after 15 or 30 minutes
of ischemia. In regions with the greatest preservation, flow values were no higher than 10% of control.
Nevertheless, compared with complete ischemia,
these extremely low flows were sufficient to retard
the decline of ATP levels (see Fig 2). The microheterogeneous pattern of NADH tissue fluorescence
seen in 4 of 5 animals after a 15-minute insult (see Fig
3B) indicates that microregions of cortex were receiving enough oxygen to keep the NAD+I
N A D H redox couple oxidized. Thus, in these regions
of low fluorescence, oxygen delivery was not alimiting
factor in energy production. This conclusion is consistent with the higher ATF and phosphocreatine levels present in brain regions that exhibited microheterogeneity compared with brains that showed a
Welsh et al: Metabolites in Diffuse Brain Ischemia 497
Tuble 3 . Efiect
011
R e x i o l d Metabolite h e l s of Recirculution following 15 or 30 Minutes oflschemiua
~
~~
~~
Region
Metabolite
Control
Recovery from
15-Min Insult
Recovery from
30-Min Insult
Lateral gyrus
ATP
PCr
Lac
NADH
2.29 2 0.02
5.40 f 0.27
0.63 f 0.14
10.5 f 1.7
1.68 f 0.04h
4.01 f 0.18
3.93 f 0.93
21.0 f 4.9
1.39 & 0.13b
3.51 f. 0.80
11.5 f 6.8
15.3 f 1.9
Medial gyms
ATP
PCr
Lac
NADH
2.33 f 0.06
5.65 2 0.14
0.69 ? 0.15
8.7 ? 1.3
1.55 4 0.03b
3.87 f 0.40
6.30 f 1.99
17.7 f 4.1
0.62 2 0.25bx
1.22 f 0.73h,c
31.9 -C 4.Bhsc
11.7 f 1.9
Sulcus
ATP
PCr
Lac
NADH
2.21 f 0.05
4.92 2 0.25
1.04 f 0.13
16.2 2 3.0
1.63 f 0.06
3.57 4 0.21
5.37 f 1.19
26.7 f 2.7
0.71 f 0.19bsc
1.07 f 0.34b*c
31.8 f 3.9bsC
13.0 f 5.1
Cingulate gyms
ATP
PCr
Lac
NADH
2.44 f 0.05
5.58 2 0.26
0.82 f 0.14
7.2 2 0.4
1.79 f 0.02b
4.55 f 0.15
4.80 f 0.45
14.3 f 2.0h
1.74 -+ 0.04b
4.22 f 0.56
9.5 f 4.2
11.7 f 0.9
White matter
ATP
PCr
Lac
NADH
2.06 f 0.04
3.11 2 0.07
0.70 2 0.17
7.9 2 0.6
1.78 f O.Obb
3.15 f 0.11
0.97 f 0.15
7.0 f 0.6
1.75
2.76
6.9
Internal capsule
ATP
PCr
Lac
NADH
1.95 2 0.05
3.45 f 0.35
0.48 f 0.07
4.6 f 0.2
1.74 5 0.02b
3.19 f 0.09
0.58 f 0.03
4.8 f 0.6
1.57 f O.OSb
3.01 2 0.15
3.3 f 0.6b*c
7.0 f 0.6b,c
Caudate nucleus
ATP
PCr
Lac
NADH
2.48 ? 0.06
6.30 f 0.40
0.86 2 0.15
12.5 f 2.0
1.83 f 0.04b
5.05 f 0.28
3.39 4 1.00
21.7 f 6.0
1.69 f 0.12b
4.20 2 0.44b
6.3 f 0.7b
34.0 f 5.3
0.02b
0.15
f 2.3b
7.0 f 0.6
f
?
"Values are mean 2 standard error. ATP, PCr, and Lac are k v e n in mmol/kg, NADH in pmoVkg. See Figure 1 for location of rekons
analyzed.
bSignificantly different from control at p < 0.05.
'Significantly different from 15-minute ischemia result a t p < 0.05.
Abbreviations same as for Table 2.
diffuse increase in fluorescence. The predominance of
a heterogeneous pattern of N A D H fluorescence at 15
minutes compared with diffuse fluorescence at 30
minutes suggests that the last remnants of blood flow
in this model were abolished between 15 and 30 minutes.
The regional pattern of N A D H fluorescence after
15 minutes of ischemia did not correspond closely to
the pattern of residual flow in the antipyrine autoradiograms [31. While the tissue fluorescence indicated patches of low NADH content ranging in size
from less than 0.1 mm to greater than 4 mm (see Fig
3B), the autoradiograms showed antipyrine only in
scattered perisulcal regions. In the deep layers of the
cortex, in white matter, and in deep gray matter, the
498 Annals of Neurology Vol 3 No 6 June 1978
optical density on the autoradiograms was not above
background. Since zero blood flow is incompatible
with low NADH, there must have been a low level of
persistent flow, below the limits of autoradiographic
detection y e t sufficient to maintain microregions of
brain in an oxidized state. T h e presence of oxidized
regions in brain with extremely low blood flow demonstrates that patchy perfusion occurs even when
ischemia is nearly complete.
In contrast to ATP levels in gray matter, white
matter ATP did not show a further decline after 15
minutes of ischemia, having reached a steady level of
1.1 to 1.3 mmoUkg (50 to 70% of control levels).
Evidently, white matter contains apool of ATP that is
extremely stable during ischemia. It is also inrerest-
Fig 6 . Recovery of corticalATP contentfollowinR90 minutes of
recircukztion after cerebralischmia lasting 15 minutes (left)or
30 minutes (right).Pour regions O f c o r t e ~wmsamplcdar illustratedin Figure I : L = hteralgyrus; S = sulcus; M = medial
gyms; C = cingubte gyms. The burs represent mean vahes
(+ SEM)for5 animalr in the no-recweq and3 animalr in the
recovery g r ~ ~ +Values
p ~ . are in mmollkg. (Asterisk indicates value signijkantb differentfrom Landlor C a t p < 0.05.)
ing that although phosphocreatine values fell comparably in white matter and gray matter, lactate and
NADH reached levels in white matter which were
only one-half those occurring in gray matter. Because
white matter has a higher lipid content and lower
water content, the intracellular space in which lactate
and NADH accumulate is presumably much smaller
than in gray matter.
The central finding of this study was the regionally
heterogeneous metabolic recovery with 90 minutes
of recirculation following a 30-minute ischemic insult. Restitution of metabolite levels was extremely
limited in discrete areas of cortex (particularly the
medial gyms and sulcus, regions M and S in Fig 1)
P i g 7. Regional variation in blood content of the brain during
ischemic recirculation. The cortex contained regions that were
extremely hyperemir (H)
and others that were blanched (B).
compared with adjacent cortical regions, white matter, or caudate nucleus. This heterogeneous pattern
of metabolic recovery is consistent with the patchy
return of cortical blood flow demonstrated in the accompanying investigation [3].
The regional pattern of metabolite impairment did
not provide a strong clue to the factors which caused
one area to recover while another did not. The clustering near the medial gyms of regions with poor
recovery suggests that the boundary zones [2] between the middle and anterior cerebral arteries may
have been preferentially affected. However, blood
flow during ischemia was reduced evenly throughout
the cerebral hemispheres, and in the postischemia
period no hypotension was present to encourage
boundary zone hypoperfusion. Alternatively, the
clustering of ischemic areas near the medial gyri may
have been a consequence of the fact that animals
were supine during the experiment, and this may
have led to intravascular stagnation and nonreperfusion that preferentially affected these lowermost cerebral gyri.
When the ischemic insult was limited to 15 minutes, partial metabolic recovery occurred in all regions analyzed. There was no evidence of heterogeneity of metabolite levels or of blood flow [3]. It is
interesting that in spite of the higher than normal
lactate levels, blood flow was only 30 to 35% of control values. The factors possibly contributing to this
situation are discussed in the accompanying report
[31.
The basis of the distinct difference in patterns of
recovery following 15 versus 30 minutes of ischemia
must reside in events initiated between the fifteenth
and thirtieth minutes of ischemia. However, the observed differences in metabolite values after these
two ischemia times was not striking; there was no
significant difference in phosphocreatine, lactate, or
NADH values for any of the regions analyzed (see
Table 2). In the case of ATP, the range of values was
much larger after 15 than after 30 minutes of ischemia, and Figure 2 strongly suggests that cortical
Welsh et al: Metabolites in Diffuse Brain Ischemia 499
ATP
NADH
ATP continued to decline between 15 and 30 minUtes of ischemia. That this continued fall of ATP during ischemia was responsible for the lack of metabolic
recovery in the animals that underwent recirculation
is doubtful, however, because after 30 minutes of
ischemia the ATP levels were as high or higher in
those regions which showed poorest restitution (the
medial gyms and sulcus, regions M and S in Fig 1).It
is possible that during recirculation, regional differences in water content, ion homeostasis, or
monoamine metabolism might govern the location of
areas of poor metabolic recovery.
Metabolic recovery from cornpkte cerebral ischemia
has been extensively investigated. Hossmann and
Kleihues 141 reported that following 60 minutes of
n o flow in the cat, cerebral ATP levels returned to
only 60% of control values within a few hours of
recirculation. However, by this time the energy
charge of the adenylate pool 111 as well as the phosphocreatine and lactate levels were nearly normal.
Similar metabolic recovery has been reported following complete ischemia in rabbits 1111 and rats 181.
The percentage reduction of the adenylate pool,
which appears to correlate directly with the length of
ischemia [8,121, may indicate the percentage of tissue that is irreversibly damaged. Alternatively, the
adenylate pool may be uniformly depressed in all the
tissue elements, and this depression need not signify
cellular damage. However, in the gerbil [7], normalization of the adenylate pool had not occurred by 24
hours following an ischemic insult that produced
patchy histological damage in the cortex 161. Thus, in
5 0 0 Annals of NeuroloRy Vol 3
N o 6 June 1978
Phosphocreatine
Lactate
Fig 8. Regional heterogeneity of metabolite leuels during recovery
from cerebral ischemia. ATP, phosphocreatine, N A D H , and
lactate were determined in various wgions of the brain slicepicturedin Figure 7 . Each value represents the mean of duplicate
assays on a sin& samplc. Values are in m d t k g for ATP.
phosphocreatine, and lactate, pnolikgfor NADH.
several models of ischemia, a persistent decrease
in ATP levels correlated with the presence of
neuropathological damage [6, 7, 10, 11, 13, 141, although the degree of neurological injury was often
minimal. In contrast to complete ischemia, severe but
incomplete ischemia may cause metabolic recovery to
be greatly impaired [12]. Thus, in rats subjected to
30 minutes of incomplete ischemia (blood flow approximately 5% of control), ATP levels recovered to
only 38% and the energy charge to 70% of control,
while lactate was fifteen times normal after 90 minutes of recirculation.
I n the present model, recovery of metabolites and
blood flow was severely impaired in focal brain regions following 30 minutes of ischemia. Whether
these abnormalities are causative factors in the production of brain damage or, rather, are the result of
prior damage cannot be inferred from the present
evidence. Nevertheless, the presence of focal areas
of persistent ischemia following a 30-minute insult is
not compatible with functional recovery. Limiting the
duration of cerebral ischemia to 15 minutes prevented focal ischemia during recirculation. However,
following 15 minutes of cerebral ischemia there was
incomplete restitution of brain ATP content and
blood flow, which may be associated with irreversible
cellular damage.
Supported by US Public Health Service Grants NS 12294-02,
NS 08803-04, and NS 10939-05, and by anEstablished Investigatorship from the American Heart Association to Dr Ginsberg, with
funds contributed in part by the Southwestern Pennsylvania Chapter.
Presented in part at the Eighth International Symposium on Cerebral Function, Metabolism and Circulation. Copenhagen, June,
1977.
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501
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