time, "lUXury perfusion" being one of the extremes. (Lassen, 1966). Several authors have raised the question of whether the changes of CBF are sec ondary to ...
JOllrnal of Cerehral Blood Flo\\' alld Metaholism 6:414-424 CD 1986 Raven Press, New York
Focal Ischemia of the Rat Brain: Autoradiographic Determination of Cerebral Glucose Utilization, Glucose Content, and Blood Flow Maiken Nedergaard,
Institute of Nellropathology and
* Medical
*
Albert Gjedde, and Nils Henrik Diemer
Physiology Department A, Panlllll Institllte, Unil'ersity of Copenhagen. Copenhagen. Denmark
Summary: Focal cerebral ischemia was induced in rats by occlusion of the middle cerebral artery. By a triple tracer technique, cerebral glucose utilization, glucose content, and blood flow were simultaneously determined. Computer-assisted autoradiography revealed a core of dense ischemia in the lateral two-thirds of the striatum. A border zone of increased 2-deoxy-o-glucose (DG) uptake surrounded the ischemic insult in the acute stage. The lumped constant was increased only moderately in the border zone. Therefore, the enhanced DG uptake re flected increased glucose consumption. CBF was re duced to 20-30% in the cortical border, while minor de-
pression and in some animals hyperemia were evident in the striate border. Six hours after the insult, the border zones of increased glucose consumption had disappeared in half the animals. In no animals examined after 20 h was glucose consumption enhanced. The study indicated a stable metabolic response to a reproducible focal insult. We conclude that continued enhancement of glucose consumption in marginally perfused areas indicates neuronal damage. Key Words: Autoradiography-Cere bral blood flow-Cerebral ischemia-2-Deoxyglucose method-Lumped constant-Middle cerebral artery oc clusion.
Clinical imaging of acute stroke requires a reli able indicator of irreversible damage and poten tially viable tissue. The visualization of CBF does not faithfully reflect the severity of ischemic damage. Positron emission tomography (PET) of the acute phase of stroke reveals a wide range of CBF values that change in either direction with time, "lUXury perfusion" being one of the extremes (Lassen, 1966). Several authors have raised the question of whether the changes of CBF are sec ondary to more fundamental changes and thus no more than an epiphenomenon (Ackerman et aI., 1981).
Quantitative 2-deoxY-D-glucose (DG) autoradiog raphy has been used in animal models of ischemia and 18F-labeled 2-fluoro-2-deoxY-D-glucose (FDG) in PET of stroke patients. Heterogeneous distur bances including increased, decreased, or normal accumulation of DG and FDG have been reported in the early phase of ischemia (Fieschi et aI. , 1978; Diemer and Siemkowicz, 1980; Wise et aI. , 1983). In experimental focal ischemia, several authors have found foci of greatly suppressed DG uptake surrounded by a rim of increased DG accumulation (Ginsberg et a\. , 1977; Welsh et aI. , 1980; Choki et aI. , 1983). In the method of Sokoloff et al. (1977), local DG accumulation is the basis for estimation of local glucose phosphorylation. However, only in normal tissue can the rate of DG be assumed to be a known fraction of glucose consumption. The "iso tope" correction ratio of DG accumulation to glu cose consumption, the lumped constant (LC), may change in conditions in which the relationship be tween plasma and brain glucose has changed.
Received August 16, 1985; accepted March 4. 1986. Address correspondence and reprint requests to Dr. M. Nedergaard at Institute of Neuropathology. Teilum Building. Frederik V's vej 11, DK 2100 Copenhagen. Denmark. Abbreviations used: DG, 2-deoxy-o-glucose; DMP. 2,2-di methoxypropane; FDG, 2-fluoro-2-deoxY-D-glucose; lAP. iodo antipyrine; LC, lumped constant; MCA. middle cerebral artery; MG, 3-0-methylglucose; PET, positron emission tomography.
414
415
MCA OCCLUSION IN THE RAT
Therefore, in pathological tissue the magnitude of the LC must be determined separately before it is possible to calculate glucose consumption from the DG accumulation. The method of Gjedde and Diemer ( 1983) allows regional, autoradiographic determination of the LC in brain regions by the simultaneous use of DG and 3-0-methylglucose (MG), a nonmetabolizable glu cose analogue. In the present study, DG and MG were used in combination with the flow tracer io doantipyrine (lAP) . Since the areas of ischemia differ with respect to both size and location in an imals with focal ischemia, only the triple-tracer technique allowed correlation of blood flow, glu cose content, and glucose consumption. The present study was designed (a) to clarify the metabolic response to focal ischemia in a stable rat model of occlusion of the middle cerebral artery (MCA) (Tamura et ai. , 198 1a); (b) to clarify the de gree to which the enhanced accumulation of DG ac tually represents increased consumption of glu cose; and (c) to relate glucose consumption to per fusion immediately after and 6 and 20 h after occlusion. MATERIALS AND METHODS Theory of glucose metabolic rate measurement The calculation of glucose metabolic rate is based on the observation that DG is phosphorylated to DG-6-P but not further metabolized or dephosphorylated to com pounds that leave the brain. Once phosphorylated, OG-6P cannot be hydrolyzed to DG because normal brain lacks significant phosphatase activity (Sokoloff et aI., 1977). After the onset of steady or near steady state, the net rate of DG phosphorylation (K) can be shown to equal the accumulated DG-6-P divided by the time-con centration integral of DG radioactivity offered to the brain by the circulation in the experimental period (Gjedde, 1982):
precursor pool is I min, while in the case of the cortical region around the infarct, it is close to 0.9 min, calculated as [lI(k2 + k3)].1 Thus, in both cases, 10 min is sufficient to ensure equilibration between the plasma and the pre cursor pool. The rate of DG-6-P accumulation is related to the glucose consumption by the relationship
CMRglc
Ca =
K
x
LC
where Ca is the plasma glucose concentration. The lumped constant is the ratio between the net rates of DG accumulation and glucose in the steady state. It can be shown to be a function of the relative amounts of glucose and DG (i.e., not DG-6-P) in the brain in the steady state (�), multiplied by the ratio between the affinities for the hexokinase reaction (1):
LC
=
�
x
II
=
I1
M*DG(x)/C*(x) a
X
MglclCa
where Mglc is the brain glucose content, M�G (x) is the steady-state radioactive DG concentration in brain, c: (x) is the steady-state radioactive DG concentration in arterial plasma, and Ca is the arterial plasma glucose con centration. In this equation, both the relative content of unphosphorylated DG in brain [M�G(x)l and the brain content of free glucose (Mglc) can be estimated with MG. MG is a non metabolizable glucose analogue, and native glucose influences the steady-state distributions of both tracer DG and tracer MG (Gjedde and Diemer, 1983). The glucose content (Mg,c) was determined as follows:
Mglc
=
Ve(Kt + Cal - Ke
where Ve is the steady-state distribution volume of MG, i.e., the ratio of tracer in brain to that in plasma at steady state; K, is the Michaelis constant of glucose transport from blood to brain: and Ke is the half-saturation content of brain glucose transport from brain to blood (Gjedde, 1982). The LC was determined from the brain glucose content by the equation
LC
=
f3
x
k*
---2.
k3
x
Ve
Ca
x
Mglc
where k; and k3 are the rate constants for the phosphory-
K (mllhg/min) total radioactivity - precursor DG activity (dpm/hg) time-plasma concentration integral (dpm
M;:'taP) - Vg c:m
f
C�dt
x
min/ml)
M;:'ta,m - Ve c�m
f
Ci,dt
I This assumption is based on calculation of the time constant x II + MeI(K, lc x g 400 flmol/hg, K,DG 3. 5, and K, lc 7 (Gjedde. 1982). In the control case, Me g 3. 2 flmol/g/min and CMR lc 125 flmol/lOO g/min. The calcu g lated values were k2 0.93 min -I . k3 0. 039 min-I, and I/(k2 + k3) 1. 03 min. In the infarct rim. Me 2. 1 flmol/g/min and CMRgk 200 flmol/lOO g/min. T he calculated values were k2 1. 07 min-I, k3 0. 095 min-I, and I/(k2 + k3) 0.858 min. In both the cases, 10 min is sufficient to ensure equilibra tion between the blood and the precursor pool. Variation of Tmax reflects variation of total regional capillary length and surface (Gjedde and Diemer, 1985). Because of the relatively well-pre served glucose content in the rim compared with low flow, it was assumed that the depressed blood flow in the infarct rim re flected a depression of the linear flow within the total capillary network and not a depression in the number of open capillaries. Thus. the Tmax value used for estimation was assumed to be at least 400 flmollhg/min in both the infarct rim and the opposite hemisphere. If Tmax were higher, the time constant of equilibra tion would be even shorter.
lI(k2 + k3) with k2 Tmax/{(K'DG x Vd) Vd)]} and k3 CMRglc/Me and where Tmax =
=
=
=
=
=
=
=
=
=
=
=
where M;ota,(D is the total tissue radioactivity derived from labeled DG, Vg is the DG "kinetic" precursor pool 2 volume equal to K , k2/(k2 + k3j , Ve is the MG distribu tion volume, and C:(t) is the radioactive DG concentra tion in arterial plasma as a function of time. At the end of 45 min, the remaining DG activity is 10% of the total ac tivity. At earlier times, labeled DG makes a significant contribution to the total activity and must be accounted for accurately (Sokoloff et aI., 1977). The equation re quires the presence of steady or near steady state. In the control case, the time constant of equilibration of the
=
=
J
=
Cereb Blood
F/0I1'
Metab. Vol. 6, No.4, 1986
416
M. NEDERGAARD ET AL.
lation of DG and glucose. respectively. As earlier de scribed. k ; /k 0.3 and r3 I (Gjedde and Diemer. 3 1983) . =
=
Experimental animals Three groups of male Wistar rats. 330-370 g. under went subtemporal craniotomy and exposure of the prox imal portion of the right MCA using a microsurgical tech nique (Tamura et al.. 1981a). The main trunk of the MCA was ligated by a 10-0 suture just proximal to the origin of the most lateral lenticulostriate branches. One group was anesthetized with Fluothane (0.75'1() until completion of the MCA occlusion. The radioactive tracers circulated during 5- 15 min after the occlusion. In the second and third groups, autoradiography was performed under halo thane anesthesia 6 and 20 h after the MCA occlusion. In these groups, the MCA was occluded during pentobar bital (50 mg/kg i.p.) anesthesia and the animals were al lowed to wake up until autoradiography. The autoradiography proceeded as follows: After tra cheostomy and endotracheal intubation. animals were ventilated by a rodent respirator with a mixture of 33'7r O2 and 66% N20 and anesthestized with 0.750 Fluothane until at least 10 min before tracer injection. Catheters were inserted into the femoral arteries and one femoral vein, and suxamethonium (75 mg/kg body weight) given. Topical Xylocaine ( 10 mg/ml) was applied to the skin in cision before withdrawal of Fluothane in all cases. Thirty microcuries [14C]DG together with 1.000 [.LCi [3H1MG was injected and circulated for 10 min. Frequent samples of blood were taken for the determination of the DG and MG plasma concentration integrals (Gjedde and Diemer. 1983) . After the last DG sample. the arterial catheters were connected to a constant-velocity withdrawal pump for mechanical integration of tracer concentration. Simul taneously, 50 [.LCi [14C]IAP was injected intravenously (Gjedde et aI., 1980) . The circulation was arrested after 20 s with KCl-Nembutal. and the brains were quickly removed and frozen in isopentane cooled to - 60°C in acetone and dry ice. Twenty-micron frozen sections were cut and for every tenth section, three neighboring sections were selected for autoradiography. Immediately after cutting, two of the three sections were dipped into 2.2-dimethoxypro pane (DMP) (2 x 1 min). DMP reacts chemically with water to form methanol and acetone. It extracts all [14C]IAP without removing DG or MG, which are insol uble in DMP (Diemer and Rosen¢rn, 1981) . The retention of DG and MG was verified by autoradiography of brains containing only one of the isotopes before and after DMP washing (data not shown) . The DMP-treated sections were exposed to x-ray films (Kodak 3H insensitive and LKB 3H and 14C sensitive). The untreated section was exposed to a 3H-insensitive film and thus indicated radio activity from both [14C]DG and [14C]IAP. The sections and 3H_ and 14C-calibrated methylmethacrylate standards were exposed to the films for 2-3 weeks. Density of the autoradiograms was measured with a Leitz TAS Plus computerized image analyzer and converted to radioac tivity by a third-degree polynomial standard curve estab lished for each film. The contents of MG and lAP were obtained by subtraction of radioactivity content in the re spective films or visualized after digitization (256 x 256 pixels) and subtraction of autoradiograms. Pseudo-color coding was performed on a Seiko D-Scan monitor inter-
J Cereb
Blood FIOlI'
Metah.
Vol. 6. No.4. 1986
faced to the image analyzer. In some brains, density was 2 determined in 50 fields (each measuring 0.1 mm ) placed along a 5-mm-long line. After evaluation of the [14CJDG film, the neighboring section from the double-label x-ray film was placed within the contours of the former autora diogram and the radioactivity corresponding to [14C]IAP content in the same 50 fields was measured and con verted to CBF values (Fig. 4). The plasma and blood sample radioactivity was deter mined in a Beckman 8000 liquid scintillation spec trometer and quench correction was performed using quench standards for both isotopes in both channels. Blood [14C]IAP activity was calculated by subtracting blood [14C]DG activity from the total 14C radioactivity.
RESULTS
Only animals in which the physiological variables remained within normal ranges during the entire course of the experiment were included in the ex perimental groups (Table 1). Figure I shows autoradiograms at the striate level in a typical experiment 15 min after MCA oc clusion. The digitized images show CMRglc, CBF, and glucose content of neighboring slices. The au to radiograms show an area of greatly depressed DG accumulation involving the lower convexity of the cortex and the lateral two-thirds segment of the striatum ipsilateral to the MCA occlusion. Perfu sion in the center of the ischemic area did not ex ceed 8 ml/ 100 g/min. A wide border of increased DG accumulation corresponded to the watershed areas between, respectively, the anterior and poste rior cerebral arteries and the MCA. The pattern of enhanced DG accumulation ex tended into the cortex (the cortical border zone) and into the medial segment of striatum and the globus pallidus (the medial border). The increase of DG accumulation in the medial border was homo geneously distributed with a sharp edge toward the septal nuclei. In the cortex, the enhancement of DG accumula tion displayed a columnar pattern perpendicular to the cortical surface, with a tendency to greater ac cumulation in the upper cortical layers (Fig. 2). The cortical border extended far more anteriorly and
TABLE 1. Physiological variables
No.
Blood pressure (mmHg)
Pco2 (mm Hg)
0
10
116 ± 8
33 ± 5
6 20
10
115 ± 7
38 ± 4
Duration after MCA occlusion (h)
Values are means
10 ±
110 ± 14
36 ± 5
Plasma glucose (mM) 9. 8 ± 2.5
10. 9 ± 2.5 9.8 ± 1.3
SE. MCA. middle cerebral artery.
417
MCA OCCLUSiON iN THE RAT
FIG. 1. Autoradiographic mapping of CMRg1c ' glucose content, CBF, and the CMRg,clCBF ratio 15 min after middle cerebral artery (MCA) occlusion. A border zone of increased glucose utilization was identified in all animals killed 15 min after MCA occlusion. In the cortical part of the border zone. perfusion was reduced to 25-30% of the level in the opposite hemisphere, while the striate border zone had higher perfusion. In the cortical rim, glucose content was reduced by 40%. In the ischemic core, no glucose was present.
especially posteriorly than the area with no DG ac cumulation. Elevation of DG accumulation was never observed outside the MCA territory. The corresponding MG autoradiograms showed an identical area of virtually no isotope in the core of the territory of the occluded artery. The LC rep resenting this area could not be calculated from the low amount of isotope. In both the cortical and the medial borders, glucose content visualized as MG autoradiograms was normal or slightly lower than in the rest of the brain. In the cortical rim, the glu cose content varied from 2. 1 f,Lmol/g initially (60% of control) to 1.9 f,Lmol/g at 6 h (66%) and 2.2 f,Lmol/ g at 20 h (69%). The LC of the border zones was increased to 0.60-0.70. Therefore, the increased
DG accumulation represented a 160-180% increase of glucose consumption when compared with the contralateral hemisphere. In the rest of the brain, glucose content was one-third of plasma glucose. The borders of increased glucose consumption related to perfusion in different ways. In the medial border, CBF was reduced to 60-75% of normal, and some animals gave evidence of hyperemia in this zone (Fig. 3). The inner part of the area of in creased DG accumulation was located above the zone of abrupt changes of perfusion. In the cortical border, perfusion was generally low, 20-30 ml/hg/ min. The perfusion displayed a pattern of gradual decline from the upper to the lower cortical layers in all animals. In some animals, the relation be-
J
eacb Blood Flo\\" Me/ab, Vol. 6, No.4. 1986
418
M. NEDERGAARD ET AL.
the low-density area. The cortical border had con tracted, but the tendency to increased DG uptake had narrowed locally into slender columns (Fig. 2). In both border zones, in addition, the enhanced DG accumulation was less intense, corresponding to an increase of the glucose consumption of no more than 130-150% of the opposite hemisphere (Fig. 4). At this time, the LC had returned to normal in the border areas. The autoradiograms from an animal 20 h after MCA occlusion are shown in Fig. 5. No border of high DG accumulation was observed in any of the animals. The flow of the striate border was normal, but the parietal border zone still revealed decreased flow (40% of normal). Twenty hours after the MCA occlusion, global CMRglc increased gradually. The abrupt changes of isotope densities \\ ne striking in the DG, MG, and lAP autoradiogram" in all groups. Isotope gradients changed up to 20-fold within a few hundred micrometers, and the zone of transition included all tracers in the anl1nal. The values of CBF and CMRglc in eight anatomi cally discrete regions ipsilateral and contralateral to the occluded MCA at 15 min and 6 and 20 h post occlusion are listed in Table 2.
FIG. 2. Printout of 2-deoxy-o-glucose (OG) autoradiograms of rats killed 15 min and 6 and 20 h after middle cerebral artery (MeA) occlusion. Serial sections at 800-f.Lm intervals are shown for respective animals. Enhanced OG accumula tion (dark) and the area of depressed isotope uptake (mask) are presented. Enhanced OG accumulation covered an ex tensive area in animals killed 15 min after the MeA occlu sion. In some animals, OG accumulation displayed a pattern of columns perpendicular to the cortical surface. In the striatum, OG accumulation was homogeneous and extended to the anatomical border of the striate border (top). Six hours after MeA occlusion, the pattern of OG accumulation in the cortex had narrowed into slender columns but cov ered the same area as above. In the striate border 6 h after MeA occlusion, increased OG accumulation was limited to a narrow rim (middle). At 20 h OG accumulation was not in creased (bottom).
tween DG accumulation and perfusion was in verted; i. e. , a high degree of DG accumulation coincided with low flow values and vice versa (Fig. 4). The threshold of perfusion coincident with in creased DG accumulation was 7 -10 ml/hg/min in both the medial and the cortical borders. The rim of enhanced glucose consumption had disappeared from half of the animals killed 6 h after the insult. In the animals with continuously in creased glucose consumption, the medial border had narrowed into a small band of a few hundred micrometers, which closely traced the margin of
J Cereb Blood Floll'
Metab, Vol. 6, No.4, 1986
DISCUSSION
The measurement of CMRglc and its correlation to CBF in the ischemic brain have special require ments. First, variations in size and location of the ischemic area necessitate simultaneous use of sev eral tracers. Second, the absence of circulatory steady state invalidates the 45-min circulation time prescribed for the DG method by Sokoloff et al. (1977). Third, normal rate constants for DG trans port into brain (K�, k;, ki) cannot be used in patho logical states. Fourth, in situations in which blood and glucose supply is limited, blood-brain barrier transport may become rate limiting for the hexoki nase reaction. DG has a higher affinity than glucose for the carrier but a lower affinity for the hexoki nase reaction. The LC equals the ratio between the rates of DG and glucose metabolism in the steady state and must increase when glucose supply is lim ited. As a consequence, the LC must be determined regionally. In an attempt to solve these problems, a triple label technique was employed using [I4C]DG, [3H]MG, and [I4 C]lAP. With three sections, the concentration of all three tracers was calculated by subtraction of autoradiograms. The use of both DG and MG allowed the determination of local glucose
MCA OCCLUSION IN TilE RAT
419
FIG. 3. Digitized, color-coded autoradiogram of CBF 15 min after middle cerebral artery occlusion in an animal with hyperemia of the globus pallidus.
content, lumped constant, and glucose phosphory lation after 10 min of tracer circulation time, rather than the usual 45 min (Siemkowicz and Gjedde, 1980; Gjedde, 1982; Gjedde and Diemer, 1983). The present study of MCA occlusion in the rat revealed an ischemic focus of severely reduced blood flow. Glucose consumption and glucose con tent reached extremely low values, and the lumped constant could not be calculated in the focus. The ischemic focus was bordered by areas of increased DG uptake in the acute stage. The glucose content of the border areas was slightly depressed and the LC only moderately in creased. Therefore, the DG accumulation repre-
sented the "true" increase of glucose consumption. MG reveals the glucose content at the blood-brain barrier, the critical site of glucose influence on the magnitude of the LC. The method does not reveal the more pro nounced hypoglycemia that may exist in the extra cellular and intracellular compartments in margin ally perfused areas with high glucose consumption. In these cases, it is the diffusion of glucose from the capillary to the site of the hexokinase reaction that may be rate limiting for the reaction, and large glu cose gradients can be expected in the tissue itself. Although the model of DG accumulation does not apply to this situation, MG actually "integrates"
J
Cereb Blood Flow Metab, Vol. 6, No.4, 1986
420
M. NEDERGAARD ET AL.
o BOUBS "..-�--------
"" �----------------- -------
-
/
6
BOUBS
i I
,', ""1,",
r"
ji
J. I
I
'··�4��--"-'-�� 20 BOUBS
'-
FIG. 4. Printouts of selected 2-deoxY-D-glucose (OG) autora diograms. Enhanced OG accumulation was detected as black areas. Regional CMRglc (f.Lmol/hg/min, solid line) and regional CBF (ml/hg/min, dotted line) were determined topo graphically in 50 fields placed along the 5-mm-long line shown in these sections, Upper two panels demonstrate the sharp delineation of the ischemic core, Note the higher level of regional CBF in the striate border compared with the cor tical border, Middle panel reveals an inverse correlation be tween regional CMRglc and CBF, Lower two panels demon strate the sharp demarcation of the ischemic core 6 and 20 h after middle cerebral artery occlusion, At 6 h a narrow rim of increased glucose utilization surrounds the ischemic focus.
these various factors, even when it does not yield a specific average tissue glucose concentration. An increase in the LC during severe ischemia was demonstrated by Ginsberg and Reivich (1979) J
Cereb Blood FIOIt' Metah, Vol, 6, No, 4, 1986
in cats. The LC returned to normal at reperfusion of the ischemic area. In humans the LC has been evaluated to be normal in lesioned regions of the brain of stroke patients, but the lesions had pro gressed to a later stage where the glucose supply adequately covered the reduced demands (Hawkins et aI., 198 1). In a comparative regional analysis of FDG and MG uptake in the brains of four stroke patients, Gjedde et al. ( 1985) tentatively identified two types of infarct: a transport-flow-limited type with low glucose content and an LC in excess of unity, and a phosphorylation-limited infarct type with nearly normal glucose content and lumped constant. The dissimilarities between the rates of perfusion of the medial and cortical border zones point to dif ferent pathophysiological bases for the increased glucose consumption. In the acute stage, the medial border zone of increased glucose consumption was located in the medial segment of the striate body and in the globus pallidus. In the medial border, the relatively high level of perfusion (in some animals the frank hyperemia) and the homogeneous eleva tion of the glucose consumption in anatomically well-defined regions do not point to an ischemic or igin of the enhanced glucose uptake. The caudate nucleus inhibits the glohus pallidus and substantia nigra by -y-aminobutyric acid-ergic projections (Kelly et aI., 1982). Hence, diminished inhibition by the ischemic part of the striatum may increase me tabolism of the efferent neurons. Metabolism was also increased in the substantia nigra, and in some instances perfusion increased ipsilateral to the MCA occlusion. The phenomenon appears to be re stricted to the early stages of cerebral ischemia and was not evident in animals examined 6 h after the occlusion. This finding is consistent with the orig inal report by Tamura et al. ( 1981 b) of hyperperfu sion in the globus pallidus and substantia nigra 30 min after the insult. The cortical border was confined to marginally perfused tissue. The enhancement of glucose con sumption persisted in about half of the animals sac rificed 6 h after the insult. In these animals, eleva tion of DG uptake was evident in the entire area of the acutely observed cortical border. Locally the increased DG uptake narrowed into thin columns perpendicular to the cortical surface, with the greatest accumulation of isotope in the upper cor tical layer (Fig. 2). In this area, the enhancement of glucose consumption may reflect anaerobic glycol ysis as a result of impaired oxygen delivery. Varying degrees of hypoxia exist in the tissue, which may explain the inhomogeneous pattern of DG uptake. Pulsinelli and Duffy (1979) demon-
MCA OCCLUSION IN THE RAT
421
CMRg1c ' glucose content, CBF, and the CMRg'c/CBF ratio 20 h after middle cerebral artery ( MCA) occlusion. No signs of increased 2-deoxy-o-glucose accumulation were found 20 h after MCA occlusion. In the cortex, the
FIG. 5. Autoradographic mapping of
pattern of glucose utilization reveals a gradual decline toward the center of ischemia, while a steeper gradient is evident in the striate border.
strated alternating columns of high and low metab olism in the cortex of hypoxic, normotensive rats. The dark columns in the autoradiograms lay be tween penetrating arteries. The authors proposed that they represented "microwatersheds" of the lowest tissue oxygen tension. To further elucidate the mechanisms behind the increased metabolism, we studied the structural damage following MeA occlusion (Nedergaard and Diemer, 1986). Outside a sharply delineated infarct, sporadic neuronal injury was evident in the cortical border. The injured neurons preferentially lay in
the upper cortical layers, and often in small groups in the neighborhood of ascending veins, probably merging with the columns of high metabolism. The noninfarcted parts of the basal ganglias were histo logically intact. Only in a rim of a few hundred mi crometers could sporadically injured neurons be traced. The narrow medial border of increased DG accumulation observed 6 h after the insult in half of the animals probably coincided with the zone of in complete neuronal injury. This zone was character ized by steep gradients between the area of isch emia and the medial portion of the striatum in J
Cereh Blood FloII' Me/ab, Vol. 6, No.4. 1986
M. NEDERGAARD ET AL.
422
TABLE 2. CBF and CMRg[c Failles in eight anatomically discrete regions Regional CBF
Right
Left
Regional CMRgI,
Glucose (f.Lmolig)
(mllhgimin) p
Left
Right
( f.Lmolihgimin)
Lumped constant
Left
p
Right
Left
Right
NS
0.49=0.15
0.44=0.10
210=28
0.62=0.21
0.45=0.09
198=28
114=20
NS
0.49=0.82
0.46=0.11
90= 13
109=18
95=
114=10
p
Oh Caudate nuclcus lat.
5=2
97=15
0.0 =0.0
3.6=0.8
Caudate nucleus med.
67=17
93= 18
3.0= 0.9
3.6=0.8
Parietal cortex
24=6
Cingulate cortcx
57= 18
Amygdalas Substantia nigra
2.1=0.7
3.5=0.7
75= 10
3.0=0.5
3.3=0.8
65= 12
85 ::!: 16
NS
104= 19
90 :':: II
NS
3.6= 1.0
3.8= O.S
2.8 =0.7
3.7=0.5
100= 20
Prefrontal cortex
37=20
99 ::!: 18
Globus pallidus
89= 20
62 :': 15
6h Caudate nucleus lat.
4 ± 2
3.4=0.7
2.9 ± 1.0
IUS :':: 8
Caudate nucleus med.
55= 18
107 ::!: 9
3.5=0.7
3.6=0.7
0.44=0.10
0=0
]()
122=20
123 ± 22
NS
0.45=0.19
0.45=0.09
NS
0.44=0.12
0.42=0.09
126=21
98=13
0.51=0.32
0.43=0.06
103=14
120= 18
NS
0.50=0.17
0.49= U.IO
120=28
94=21
0=0
137 :': 23
60=28
132 :':: 19
0.2=0.5
2.9=0.5
2.7=0.7
3.0=0.8
NS
0.55=0.14
2.6 ::!: 0.6
0.70=0.18
0.52 :':: 0.13
63=30
120=21
2.8=0.7
NS
0.56=0.13
0.53=0.13
107=23
125 :':: 21
!'IS
2.6=0.4
2.5=0.6
NS
0.56=0.09
0.58=0.14
94=21
NS
3.0 :':: 0.6
NS
0.50= 0.08
(1.51 :': 0.10
108 ::!: 16
103= 15 110=17
NS
1.9 ::!: 0.5
2.9 ::!: U.7
2.1=5.4
0.52 :':: 0.09
0.51 ::!: U.14
Parietal cortex
30= 6
110= 10
Cingulate cortex
55= 6
78= 16
Amygdalas
75=12
94 ::!: 10
Substantia nigra
73=11
95=17
3.1 ± 0.5
Prefrontal cortex
51= 8
98 =II
2.6 :':: 0.8
3.0 :': 1.0
NS
0.56=0.17
0.51 :':: 0.17
95 :': 26
137=23
Globus pallidus
56= 4
63 = 12
NS
2.7 :':: 0.8
2.8 :': 0.7
NS
0.55 :':: 0.16
0.53 :':: 0.13
104 :':: 27
102= 18
0.5=0.7
2.7 ::!: 0.5
3.1=0.6 3.0 ::!: 0.5
2.1 :': 2.9
0.48=0.09
NS
NS
0.52 :':: 0.1
0.49 :':: 0.08
3.2=0.7
NS
0.60=0.25
0.47 :':: 0.10
51 ± 25
20 h
NS
99 ::!: 13
NS
NS
Caudate nucleus lat.
10= 7
Caudate nucleus med.
88= 28
101= 10
Parietal cortex
41= 14
100 :':: 9
2.2 ::!: 0.9
Cingulate cortex
74= 18
98= 7
2.9=0.8
NS
0.51= 0.09
0.50 :':: 0.14
102= 22
127= 23
Amygdalas
78= 10
85 ::!: 12
2.8 :':: 0.5 2.7=0.6
2.7=0.6
NS
0.52 :':: 0.12
0.53=0.12
100= 18
100= 14
NS
Substantia nigra
80= 9
90 :':: 5
3.0 =0.8
2.8 =0.6
NS
0.49=0.13
0.51=0.11
101=16
95=27
NS b
Prefrontal cortex
47= 12
100 :':: 14
Globus pallidus
59= 15
68 :':: 13
NS
NS
0=0
58 ::!: 18
2.9= 0.4
2.8 =0.5
NS
0.50=0.07
0.51=0.09
93=21
2.5=0.8
2.6=0.7
NS
0.55=0.18
0.54=0.15
87=14
147= 15 146 :':: 24 121= 16
120 ::!: 20 95 ::!: 10
NS
Values are means= SE.
By Student's t test. "significantly different at p < 0.001; bsignificantly dilferent at p < 0.05; 'signilicantly different at p < (Ull.
which CBF was relatively preserved. Also, in this zone of transitions, the elevated DG uptake may reflect anaerobic glycolysis. Severe lactacidosis is certain to develop locally and may cause the neu ronal injury (Siesj6, 1984). After the ischemic insult, in studies of global ischemia in rats, Diemer and Siemkowicz ( 1980) noted that the CAl region of the hippocampus, the globus pallidus, the substantia nigra, and the cere bellar molecular layer develop the same degree of increased glucose uptake and subsequent selective neuron loss. In very early reperfusion, global isch emia was followed by global hyperemia. For this reason, the increased metabolism in the vulnerable regions is probably not an effect of low oxygen supply (Pasteur effect). Instead, it is tempting to speculate that the enhanced glucose utilization may be a manifestation of neuronal repair. The coinci dence of high metabolism and dead neurons sup ports this explanation. Perfusion below the threshold of ischemia causes neuronal membranes to leak potassium and neuro transmitters (Astrup et aI., 1977; Siesj6, 1978). In vitro experiments show that glial cells take up po tassium and neurotransmitters (glutamate and "1aminobutyric acid) in association with a drastic in crease of their metabolism (Hertz, 1981), thus acting as tissue "buffers." In the marginally per-
J
Cereb Blood Floii' Metab, Vol. 6, No. 4, 1986
fused areas in cortex and in the narrow zone of steep perfusion gradients in the striatum, some neu rons fall under the threshold of "membrane failure" and leak ions. The more ischemia-resistant astrocytes may outlive the injured neurons and, in response to the disturbance of the fluid environ ment in the extracellular space, may increase their glucose consumption. As an alternative to the hypothesis of local potas sium efflux, we propose that the marked leakage of potassium in the ischemic core may cause waves of potassium to propagate to the border areas. Potas sium transients are related to the phenomenon of spreading depression. Spreading depression is as sociated with increased glucose consumption (Shinohara et aI., 1979; Gjedde et aI., 1981) and spreads preferentially in the upper cortical layers (Leao, 1951). In the marginally perfused borders of focal ischemia, failure of potassium clearance may prolong the otherwise transient elevations of the extracellular potassium. Potassium leakage usually leads to calcium entry into cells, and intracellular accumulation of calcium is widely suspected to be involved in the induction of irreversible ischemic cell damage (Siesj6, 1983). The increase of DG uptake in the border zone of infarcts was originally reported by Ginsberg et al. (1977) in cats with MCA occlusion and was later
423
MCA OCCLUSION IN THE RAT
confirmed by several groups (Schwartzman et a!. , 1978; Welsh et a!. , 1980; Choki et a!. , 1983; Shigeno et a!. , 1983). Choki et al. ( 1983) measured CBF and DG uptake by a double-label autoradiographic method. The DG accumulation was found to be constant over a wide range of flow values, until it in a stepwise manner increased below a threshold of 20-40 mllhg/min in awake cats and 5- 12 mllhg/min in pentobarbital-anesthetized cats. In rats, we found a low threshold of 7 - 10 mllhg/min, while th ere was no correlation to CMRglc at higher values. The divergence probably reflects the dif ferent time frames of MCA occlusion and autoradi ography. Choki et al. performed the autoradiog raphy 2 h after the MCA occlusion. The effect of diminished inhibition from the ischemic caudate nucleus to the medial border may have ceased at that time, leaving only the elevated glucose con sumption in the marginally perfused areas. Welsh et al. ( 1980) found that increased DG uptake in cortex coincided with a wide range of ATP and phospho creatine contents in tissue samples. However, the inhomogeneous pattern of DG accumulation may render sampling of tissue with exclusively high or low DG uptake impossible. A human parallel to the findings in rats was dem onstrated by Wise et al. ( 1983), who visualized re gional CBF, oxygen extraction, and oxygen metab olism by PET in patients after acute stroke. The pa tients studied shortly after the onset of stroke ( 1.5-24 h) had elevated oxygen extraction with preserved oxygen metabolism relative to blood flow to the infarct. Later, significantly depressed glucose metabolism was observed in association with either "luxury perfusion" or decreased perfu sion. Within the resolution of PET, no rim of per sistently elevated oxygen extraction was identified around the edge of the infarct. The present investigation demonstrated a highly predictable pattern of metabolic response to MCA occlusion. Border areas of increased glucose con sumption were noticed acutely in all animals, with only minor variations of the location. The DG ac cumulation was not associated with major changes of the LC because the glucose content in the tissue was reduced only slightly. An effect of low oxygen supply with the trickle of flow to the cortical border cannot be excluded (Pasteur effect). In the medial border where CBF was close to normal, the in creased metabolism may reflect increased func tional demands. Future studies must search for a correlation between the acute metabolic alterations and later extension of the infarct. This correlation is particularly relevant to the interpretation of images of blood flow and metabolism obtained as a �
guide to therapeutic interventions in the acute phase of stroke.
REFERENCES
Ackerman RH, Alpert NM, Correia JA, Finkelstein S, Buo nanno F, Davis SM, Chang JY, Brownell GL, Taveras JM (1981) Importance of monitoring metabolic function in as sessing the severity of a stroke insult (CBF: an epiphe nomenon'n. J Cereh Blood Flow Metah l(suppl 1):502-503 Astrup J, Symon L, Branston NM, Lassen NA (1977) Cortical evoked potential and extracellular K+ and H+ at critical levels of brain ischemia. Stroke 8:51-57 Choki J, Greenberg JH, Jones SC, Reivich M (1983) Correlation between blood flow and glucose metabolism in focal cere bral ischemia in the cat. J Cereh Blood Flow Metah 3(suppl 1):399-400 Diemer NH, Rosen¢rn J (1981) Determination of local cerebral blood flow and glucose metabolism or transfer by means of a double autoradiographic method. J Cereh Blood Flow Metah I(suppl 1):72-73 Diemer NH, Siemkowicz E (1980) Increased 2-deoxyglucose uptake in hippocampus, globus pallidus and substantia nigra after cerebral ischemia. Acta Nelll'ol Scand 61:56-63 Fieschi C, Sakurada 0, Sokoloff L (1978) Local cerebral glucose utilization during resolution of embolic experimental isch emia. In: Advances in NearologJ'. New York, Raven Press, pp 223-229 Ginsberg MD, Reivich M (1979) Use of the 2-deoxyglucose method of local cerebral glucose utilization in the abnormal brain: evaluation of the lumped constant during ischemia. Acta Nellrol Scand 60(suppl 72):226-227 Ginsberg MD, Reivich M, Giandomenico M, Greenberg JH (1977) Local glucose utilization in acute focal cerebral isch emia; local dysmetabolism and diascisis. Neurology 27:1042-1048 Gjedde A (1982) Calculation of cerebral glucose phosphorylation from brain uptake of glucose analogs in vivo: a re-examina tion. Brain Res ReI' 4:237-274 Gjedde A, Diemer NH (1983) Autoradiographic determination of regional brain glucose content. J Cereh Blood Flow Metah 3:303-310 Gjedde A, Diemer NH (1985) Double-tracer study of the fine regional blood-brain glucose transfer in the rat by com puter-assisted autoradiography. J Cereh Blood Flow Metah 5:282-289 Gjedde A, Hansen AJ, Siemkowicz E (1980) Rapid simultaneous determination of regional blood flow and blood-brain glu cose transfer in brain of rat. Acta Physiol Scand 108:321330 Gjedde A, Hansen AJ, Quistorff B (1981) Blood-brain glucose transfer in spreading depression. J Nellrochem 37:807-812 Gjedde A, Wienhard K, Heiss W-D, Kloster G, Diemer NH, Herholz K, Pawlik G (1985) Comparative regional analysis of 2-fluorodeoxyglucose and methylglucose uptake in brain of four stroke patients. With special reference to the re gional estimation of the lumped constant. J Cereh Blood Flow Metah 5:163-178 Hawkins AR, Phelps EM, Huang S, Kuhl DE (1981) Effect of ischemia on quantification of local cerebral glucose meta bolic rate in man. J Cereh Blood Flow Metah 1:37-51 Hertz L (1981) Features of astrocytic function apparently in volved in the response of central nervous tissue to ischemia -hypoxia. J Cereh Blood Flow Metah I:143-153 Kelly PAT, Graham Dl, McCulloch J (1982) Specific alternations in local cerebral glucose utilization following striatal lesions. Brain Res 233:157-172 Lassen NA (1966) T he luxury-perfusion syndrome and its pos sible relation to acute metabolic acidosis localised within the brain. Lancet 1:1113-1115 Leao AAP (1951) T he slow voltage variations of cortical
J
Cereh
Blood Flow Metah, Vol. 6, No.4, 1986
M. NEDERGAARD ET AL.
424
spreading depression of activity.
Electroencephalogr Clin
Nedergaard M, Diemer NH (1986) Sporadic neuronal necrosis in the peri-infarct zone following MCA occlusion in the rat. Acta Neurol Scand 73:87
Pulsinelli WA, Duffy TE (1979) Local cerebral glucose metabo lism during controlled hypoxemia in rats. Science 204:626-
Experientia 34:1175-1176
Shigeno T, McCulloch J, Kirkham 0, Mendelow 0, Graham Dl (1983) Ischemic core and penumbra: A multifactorial anal ysis of the metabolic consequences. J Cereh Blood FloI\' Metah 3(suppl 1):403-404 Shinohara M, Dollinger B, Brown G, Rapoport S, Sokoloff L (1979) Cerebral glucose utilization: local changes during and after recovery from spreading cortical depression. Science 203:188-190
Siemkowicz E, Gjedde A (1980) Post-ischemic coma in rat: ef fect of different pre-ischemic blood glucose levels on cere bral metabolic recovery after ischemia. Acta Physiol Scand 110:225-232
Siesj6 BK (1978) Brain Energy Wiley & Sons, pp 398-446
Cereb
J New'o
Sokoloff L, Reivich M, Kennedy C, Des Rosiers MH, Patlak CS, Pettigrew KO, Sakurada 0, Shinohara M (1977) The (14C) deoxyglucose method for measurement of local cere bral glucose utilization: Theory, procedure, and normal values in the conscious and anesthetized albino rat. J Neu rochem 28:897-916
629
Schwartzman RJ, Kogure K, Busto R (1978) T he sequential uptake of (14C) deoxyglucose in brain after embolic stroke.
J
Siesjii BK (1984) Cerebral circulation and metabolism. sllrg 60:883-908
NeurophysioI3:3l5-32l
Blood Flow
Metab,
Metaholism.
Vol. 6, No.4,
New York: John
Tamura A, Graham 01, McCulloch J, Teasdale GM (1981a) Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J Cereh Blood FloI\' Metah 1 :53-60 Tamura A, Graham Dl, McCulloch J, Teasdale GM (1981h) Focal cerebral ischaemia in the rat: 2. Regional cerebral blood tlow determined by I14C] iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereh Blood Flow Metah 1:61-68
Welsh FA, Greenberg JH, Jones SC, Ginsberg MD, Reivich M (1980) Correlation between glucose utilization and metabolic levels during focal ischemia in cat brain. Stroke 11:79-84 Wise RJS. Rhodes CG. Gibbs JM, Hatazawa J, Palmer T. Frackowiak RSJ. Jones T (1983) Disturbance of oxidative metabolism of glucose in recent human cerebral infarct. Ann Neural 14:627-637
1986
