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(Stroke. 1995;26:2112-2119.)
© 1995 American Heart Association, Inc.

Articles

Sequential Studies of Severely Hypometabolic Tissue Volumes After Permanent Middle Cerebral Artery Occlusion

A Positron Emission Tomographic Investigation in Anesthetized Baboons

Omar Touzani, MSc; Alan R. Young, PhD; Jean-Michel Derlon, MD; Vincent Beaudouin; Gilles Marchal, MD; Patrice Rioux, MD, PhD; Florence Mézenge; Jean-Claude Baron, MD Eric T. MacKenzie, PhD

From Cyceron, Biomedical Cyclotron Unit of Caen, University of Caen CNRS URA 1829, INSERM U320, CEA DSV/DRIPP, and University Hospital of Caen (France).

Correspondence to Omar Touzani, Cyceron (CNRS URA 1829), Boulevard Henri Becquerel, BP 5229, 14074 Caen Cedex, France.

	Abstract

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Background and Purpose In the positron emission tomography literature, markedly hypometabolic brain tissue (oxygen metabolism <1.3 to 1.7 mL · 100 g^-1 · min^-1) has often been equated with irreversible damage in the human brain. By serial positron emission tomography measurements, we investigated the temporal evolution of the volume of severely hypometabolic brain tissue after permanent middle cerebral artery occlusion in anesthetized baboons with, as a perspective, the development of rational therapeutic strategies.

Methods Seven anesthetized and ventilated baboons underwent sequential positron emission tomography examinations with the ¹⁵O steady-state technique before and 1, 4, 7, and 24 hours and 14 to 29 days after occlusion. In each baboon the infarct volume was calculated by quantitative histological procedures after 19 to 41 days of occlusion.

Results The sequential measurement of regional oxygen metabolism demonstrated an extension (for 24 hours) of the volume of severely hypometabolic tissue as defined by both absolute and relative metabolic thresholds, and this profile of evolutivity is observed no matter the threshold used. Mean (±SEM) infarction volume of 2.4±0.6 cm³ was comparable to a tissue volume with oxygen consumption <40% of contralateral metabolism. The volume of hypometabolic tissue was essentially stable at the 1-, 4-, and 7-hour postocclusion studies, increased markedly at the 24-hour study point, and increased even further in the chronic-stage study (on average, 17 days after occlusion). The tissue that eventually displayed a severely hypometabolic state at the final measurement showed a significant decrease of oxygen metabolism and cerebral blood flow at each time analyzed. In that tissue, the oxygen extraction fraction increased significantly at 1 hour (although not thereafter).

Conclusions The extension of severely hypometabolic volume after middle cerebral artery occlusion reinforces the concept of a dynamic penumbra and suggests the existence of a relatively large window of therapeutic opportunity in which it may be possible to develop neuroprotective strategies. Our study suggests that maximum infarct volume is determined at some time between 24 hours and 17 days after permanent middle cerebral artery occlusion in anesthetized baboons.

Key Words: cerebral blood flow • neuroprotection • tomography, emission-computed • baboons

	Introduction

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A growing body of evidence indicates that the extent of brain damage continues to increase with time after the experimental occlusion of a major cerebral artery. During the last decade several small animal models of focal cerebral ischemia have been developed to assess the temporal evolution of infarct size (assessed by postmortem neuropathology) after reversible or permanent arterial occlusion.^{1 2 3 4} Although the knowledge of temporal thresholds for infarction and its evolution is important for the development of therapeutic strategies, until now no study has addressed the issue of this evolution in the same subject. This lack of sequential analysis has consequently given rise to data with a large interindividual variation.

The feasibility of performing serial PET studies in the nonhuman primate subjected to MCAO has already been demonstrated.^{5 6 7} When undertaken in both the acute and chronic stages, sequential studies in the same animal should provide detailed information on the hemodynamic and metabolic state of ischemic tissue that will evolve toward necrosis.⁸

The goal of the present investigation was to study the temporal evolution of the volume of severely hypometabolic tissue (based on repeated PET measurements) and its pathophysiological characteristics after permanent MCAO in the anesthetized baboon. A better understanding of the temporospatial evolution of this volume after stroke may be of value for the evaluation of an eventual therapeutic intervention. Our rationale was that below an undetermined level of oxidative metabolism, neuronal viability would be impossible. In the PET literature, previous studies have attempted to address the issue of the metabolic threshold that is necessary to maintain the structural integrity of cerebral tissue after stroke in humans.^{9 10 11 12} These studies have suggested that brain regions with a CMRO₂ <1.3 to 1.7 mL · 100 g^-1 · min^-1 are often equated with irreversible damage.

	Materials and Methods

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Anesthesia and Physiological Monitoring
The studies were performed on seven adolescent male baboons (Papio anubis; weight, 11 to 14 kg). The baboons were fasted for 24 hours before the study and initially sedated with the ultrashort-acting barbiturate methohexital (20 mg/kg IM; Brietal); anesthesia was induced by the administration of etomidate (3 mg/kg IV; Hypnomidate) and clonidine (80 µg, infused intravenously over 10 minutes; Catapressan). After muscular relaxation with atracurium (0.5 mg · kg^-1 IV, and thereafter 0.75 mg · kg^-1 · h^-1; Tracrium), endotracheal intubation was performed, and artificial ventilation was adjusted to maintain normocapnia. Anesthesia was maintained with nitrous oxide in oxygen (N₂O/O₂=2:1) and etomidate (0.3 to 0.6 mg · kg^-1 · h^-1). This anesthetic regimen, based on agents with short plasma half-life periods, was chosen to minimize cerebral metabolic depression and to facilitate a rapid recovery.¹³ Via percutaneous femoral cannulas, arterial samples were withdrawn periodically for the measurement of PaCO₂, PaO₂, pH, hemoglobin, hematocrit, and plasma glucose levels. Body temperature was maintained within normal limits by heating blankets. Total fluid balance was controlled as followed: inspired gases were humidified at 38°C; replacement therapy was achieved with saline and/or Ringer's solution at a rate of approximately 4 mL · kg^-1 · h^-1, and urine output was monitored. Serum electrolytes were periodically measured. Cardiovascular parameters (electrocardiogram, heart rate, and arterial pressure) were monitored continuously. At the end of each PET study, cephamandol (45 mg · kg^-1 IM; Kefandol) was administered, and the baboon was allowed to recover fully and returned to its cage. Five hours after occlusion, each baboon received a transfusion of 200 mL of concentrated human erythrocytes; the transfusion was preceded by the administration of dexamethasone (2 mg IM; Soludecadron).

MCAO
Under aseptic conditions and anesthesia supplemented with isoflurane (0.5% to 1.5%; Forene), which was discontinued at least 2 hours before the first PET examination, the right MCA was exposed by a transorbital approach under the operating microscope.¹⁴ Because of the extremely abundant leptomeningeal anastomoses that could lead to variable pathological outcomes,^{15 16} two microvascular clips were placed permanently, one on the proximal part of the main MCA trunk and the other on the orbitofrontal branch. The reconstruction of the dura mater and orbit followed by a tarsorrhaphy allowed recovery without complications in all baboons and permitted long-term survival. After recovery from anesthesia, the baboons were returned to their cages and given access to water and fruits, then observed for 2 to 4 hours and daily. Usually the day after occlusion the baboons were able to remain seated, to move, and to feed without assistance despite a contralateral hemiparesis (which disappeared progressively within the following 2 weeks).

PET Examination Protocol
For the assessment of local CBF, CMRO₂, OEF, and CBV, we used the ¹⁵O steady-state technique¹⁷ with correction for intravascular tracer with C¹⁵O and a seven-slice LETI TTV03 high-resolution (with intrinsic resolution for ¹⁵O: 7.0x7.0x9.0 mm; coordinates x, y, and z) PET camera (CEN).^{18 19 20} Special care was taken to ensure a stable gas flow delivery; all arterial ¹⁵O measurements were based on three samples, each taken in duplicate. Each of the seven baboons underwent serial examinations: one control session approximately 2 weeks before MCAO and thereafter at 1, 4, 7, and 24 hours and 14 to 29 days after MCAO. Two of these baboons also underwent two examinations in the chronic stage (one at 7 days and then at 22 days, the other at 14 days and then at 21 days after occlusion, although only the first of repeated chronic studies was used for statistical analysis). Thus, n=7 at control, 1, 7, and 24 hours and 17 days (on average) after MCAO, whereas n=6 at 4 hours after MCAO, where n is the number of PET studies based on seven baboons (because of technical failure, one study could not be performed). Two additional baboons were discarded for reasons of technical failure. In each PET study, to obtain reproducible (intra-animal and interanimal) head positioning within the camera tunnel, the baboon's head was fixed in a specially designed frame by means of ear bars placed in the bony portion of the external auditory canal; the positioning was checked by a radiogram. This procedure allowed optimal repositioning of the animal's head within the camera tunnel in all sequential PET sessions. Seven planes (-27 mm to +45 mm parallel to the canthomeatal line) were imaged according to an anatomic PET atlas.²¹ Before each PET session, a ⁶⁸Ga-⁶⁸Ge transmission scan was performed.

Data Analysis
PET data were globally analyzed by an objective method developed in our laboratory, based on functional quantitative thresholds.^{22 23} To reduce data size and improve statistical sampling, all parametric images (1x1-mm pixel) were transformed onto a 4x4-mm grid, in which each 4x4-mm voxel is a region of interest identified by its coordinates x, y, and z; based on the control CMRO₂ images, the contour of the brain was outlined on three planes (+9 mm, +21 mm, and +33 mm parallel to the canthomeatal line) that included most of the MCA territory. Then all 4x4-mm voxels with CMRO₂ values below a given threshold were identified by computer in each plane. The volume of hypometabolic tissue was then calculated by summation in each slice, interpolated between slices (interslice distance, 3 mm), and extrapolated to those slices in which no hypometabolic tissue was identified. This PET image analysis technique (with realignment, if necessary, of the brain isocontours) allows one to follow each selected voxel for all the measured parameters as a function of time.

Based on the available data in the literature obtained from clinical studies^{9 10 11 12} and in the absence of metabolic thresholds for irreversible damage in the nonhuman primate, we used a metabolic threshold of 1.5 mL · 100 g^-1 · min^-1 to characterize severely hypometabolic tissue. Based on the results obtained with this absolute threshold, we subsequently chose to test normalized metabolic thresholds (40%, 45%, and 50% of contralateral metabolism).

To analyze the pathophysiological characteristics of tissue volume not yet severely hypometabolic but that will evolve toward this state at the final examination, we subtracted those pixels with CMRO₂ <45% of contralateral CMRO₂ at 1, 7, and 24 hours from the total volume of defined pixels found in the chronic-stage study.

Neuropathology
Nineteen to 41 days after MCAO, the baboons were submitted to an MRI examination to localize the lesion and were then deeply anesthetized; the brains were fixed in situ by transcardiac perfusion with a solution of FAM (formaldehyde 40%, glacial acetic acid, and absolute methanol in the ratio 1:1:8). Thereafter, the brains were removed and placed in the FAM fixative for a minimum of 7 days. Subsequently, the extremities of the frontal lobes, brain stem, and cerebellum were cut, and the remaining block was embedded in paraffin. Coronal sections 15 µm thick were cut throughout the rostrocaudal extent of the brain; the sections were then stained by hematoxylin and eosin. For measurement of infarct volume, 10 equidistant coronal slices covering the entire lesion were chosen,²⁴ and the infarcted surface (the difference between the contralateral hemisphere area and ipsilateral noninfarcted area) was measured in each slice by an image analyzer (BIOCOM RAG 200). The ventricular spaces were subtracted from both hemispheres. The total histological volume of infarction was calculated by integration of the areas over 10 equidistant sections and the distance between them.

Statistical Analysis
All data are expressed as mean±SEM. The physiological parameters at each time point were compared by one-way ANOVA. The temporal evolution of hypometabolic volumes (for all the thresholds used) was analyzed by ANOVA with repeated measurements. For the characteristics of tissue that will evolve toward a severely hypometabolic state, the side-to-side difference was compared by Student's paired t test. Statistical significance was set at P<.05.

	Results

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Physiological Data (Table 1)
Mean arterial pressure, heart rate, PaO₂, PaCO₂, pH, hematocrit, and body temperature remained remarkably stable during the sequential PET studies. However, the hemoglobin concentration showed a significant decrease (ANOVA; P=.001 compared with pre-MCAO values) at the latest measurement. The decrease in hemoglobin concentration may be explained by the repeated use of etomidate.²⁵ Moreover, a moderate but significant increase in plasma glucose concentrations was noted at 4 and 7 hours after MCAO (ANOVA; P=.002).

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters Before and After MCAO in Seven Anesthetized Baboons

Neurological Deficit
In all baboons, MCAO caused a neurological deficit that became observable in the hour after recovery from anesthesia (8 hours after occlusion); all animals presented with a contralateral hemiplegia associated with stupor and deviation of the eye and head toward the ipsilateral side. This neurological deficit recovered progressively, and after 2 weeks all baboons (except one, in which the neurological state deteriorated after a severe hemolysis) showed a nearly complete functional recovery.

Evolution of Metabolic Volume With CMRO₂ <1.5 mL · 100 g^-1 · min^-1
Serial images for CMRO₂ obtained in the same baboon at various times after occlusion showed an enlargement of the hypometabolic region with time (Fig 1). A marked impairment of oxidative metabolism was noted essentially in the deep MCA territory in the acute stage and extended laterally and posteriorly with time. A not dissimilar profile was seen in the CBF images, although the defect in CBF in the acute stage was larger than that of CMRO₂ (Fig 2).

View larger version (0K): [in this window] [in a new window]   Figure 1. Parametric PET images of CMRO2 obtained from a single plane (parallel to and 21 mm above the canthomeatal line) in one anesthetized baboon before and at various times after right MCAO show a progressive expansion of a markedly hypometabolic zone that extends laterally and posteriorly from the deep MCA territory. The images are displayed with the anterior up and the right side of brain to the left; the pseudocolor representation is shown according to the scale depicted on the right.

View larger version (0K): [in this window] [in a new window]   Figure 2. Parametric PET images of CBF obtained in the same baboon as in Fig 1. These sequential images show the same profile of progressive extension of hypoperfused region occurring in the MCA territory. See legend to Fig 1 for further details.

Initially, to define severely hypometabolic tissue and based on available data in the literature, we chose a metabolic threshold of 1.5 mL · 100 g^-1 · min^-1. The volume of tissue with CMRO₂ below this value is expressed as a percentage of the contralateral hemisphere volume (Table 2). In all baboons, baseline studies failed to reveal voxels with CMRO₂ <1.5 mL · 100 g^-1 · min^-1. The volume of severely hypometabolic tissue increases with time (a repeated ANOVA showed a significant time effect, P=.002). Nevertheless, the volume measured by this procedure 17 days (on average) after occlusion (32%=13.1 cm³) was considerably greater than the values published from a not dissimilar model,²⁶ as well as our own histological data, which will be discussed below.

View this table: [in this window] [in a new window]   Table 2. Evolution of Hemispheric Volume With CMRO2 <1.5 mL · 100 g-1 · min-1

Evolution of Hypometabolic Tissue Volumes Defined as a Percentage of Contralateral Hemispheric Metabolism
Based on results of the initial analyses discussed above and on the fact that the metabolic threshold may not be constant throughout the time course of ischemia, we then used a relative metabolic threshold (which would take into account a possible alteration in global metabolism²⁷ ) to characterize severely hypometabolic tissue. We chose to test thresholds of 40%, 45%, and 50% of contralateral CMRO₂ (Table 3). None of the baboons presented with regions of interests (4x4-mm voxels) with a CMRO₂ below these thresholds before occlusion. The tissue volume with CMRO₂ <45% of contralateral CMRO₂ increased with time and quadrupled between 1 hour and 24 hours and doubled between 24 hours and the final PET measurement at 17 days (on average). A repeated-measures ANOVA showed a significant time effect (P=.0001) and significant difference between severely hypometabolic volumes at 1 hour and 24 hours as well as between 1 hour and 17 days after occlusion (P<.03) but only a tendency for these volumes to increase between 1 hour and 7 hours (P=.119). One notes that a similar profile of evolutivity is seen no matter the threshold used, and as expected the volume of severely hypometabolic tissue increases with the threshold used.

View this table: [in this window] [in a new window]   Table 3. Evolution of Tissue Volumes Corresponding to Three Metabolic Thresholds

Quantitative Histology
To validate the choice of a metabolic threshold that would define irreversibly damaged tissue under our experimental and imaging conditions, we used quantitative neuropathology. Low-level light microscopy of the stained tissue sections revealed a sharply demarcated infarction that embraced the putamen, caudate nucleus, and internal and external capsules; in two baboons the parasylvian cortex was involved. Based on 10 equidistant coronal sections, the volume of infarct was 2.4±0.6 cm³ (mean±SEM). This volume, determined histologically, is comparable to the tissue volume with CMRO₂ <40% of contralateral CMRO₂ (2.9±0.6 cm³, mean±SEM) and correlates with both tissue volumes with CMRO₂ values <45% (as measured at 17 days after occlusion) (4.3±0.9 cm³) and <50% (5.6±1.1 cm³) of contralateral CMRO₂ (Table 4). By correlation analysis, where y is the hypometabolic volume and x the histological volume of infarction, the following Pearson correlation coefficients were found: (1) at CMRO₂ <45%, r=.89 (P<.05) and y=1.4x+0.90; (2) at CMRO₂ <50%, r=.83 (P<.05) and y=1.6x+1.79.

View this table: [in this window] [in a new window]   Table 4. Histological Infarct Volume and Metabolic PET Volume Measured 17 days After Occlusion

Characteristics of Tissue That Evolves Toward a Severely Hypometabolic State
As a first approach to analyze the pathophysiological characteristics of tissue that is not yet severely hypometabolic but that will evolve toward this state at the final examination, we subtracted those pixels with CMRO₂ <45% of contralateral CMRO₂ at 1, 7, and 24 hours from the same at the chronic study. The physiological parameters of this tissue obtained by subtraction (with, by definition, CMRO₂ values >45% of contralateral CMRO₂) were then compared with contralateral mirror pixels (Table 5). Paired t tests demonstrated a significant (P<.05) decrease, relative to the contralateral hemisphere, of both CMRO₂ and CBF at each time analyzed. The OEF showed a significant increase (P<.05) at 1 hour after occlusion but only a slight increase from contralateral tissue (not significant) at 7 hours and no interhemispheric difference at 24 hours. The percent changes are shown in Fig 3. CBV measured in this threatened tissue was not different from contralateral tissue at all times analyzed.

View this table: [in this window] [in a new window]   Table 5. Characteristics of Tissue That Evolves Toward a Severely Hypometabolic State

View larger version (30K): [in this window] [in a new window]   Figure 3. Bar graphs show characteristics of tissue that evolves toward a severely hypometabolic state. (All parameters are expressed as percent difference from contralateral mirror tissue.) This tissue volume was obtained by subtraction of pixels with CMRO2 <45% of contralateral CMRO2 at 1, 7, and 24 hours from those at the last PET measurement. Each value represents mean (±SEM) of seven baboons. *Significant side-to-side difference (P<.05) by Student's paired t test.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we investigated the temporal evolution of the volume of severely hypometabolic tissue in the anesthetized baboon subjected to permanent MCAO. Our findings were based on the analysis of regional metabolic data, obtained from sequential PET examinations, in both the acute and the chronic stages of focal cerebral ischemia. Previously, Pappata et al⁶ reported a significant decline in CMRO₂ at 4 hours compared with pre-MCAO values in baboons. This decrease only affected the deep MCA territory. Since chronic studies were not attempted, the final tissue outcome was not determined. Further studies in the cat have been published recently to show the pathophysiological evolution of ischemic tissue in the 24 hours after MCAO.⁸

CMRO₂ Thresholds for Ischemia in Humans
Because of technical limitations in the measurement of regional CMRO₂ in small animals, several authors have simply addressed the question of the flow thresholds that would eventually result in structural damage of brain tissue after MCAO.^{28 29 30 31} However, in stroke patients certain PET studies have investigated the level of oxidative metabolism below which irreversible brain damage occurs. Lenzi et al³² reported (in 15 patients in whom morphological imaging was not used, studied between 1 and 34 days from the onset of symptoms) that a metabolic threshold of 1.25 mL · 100 g^-1 · min^-1 (or 40% of the contralateral values) correlated with a poor clinical outcome. Baron et al⁹ showed that a CMRO₂ threshold >1.7 mL · 100 g^-1 · min^-1 separated viable tissue from tissue that was already or would ultimately become infarcted below this threshold (as validated by chronic-stage structural imaging). Furthermore, Powers and colleagues¹⁰ found that 80% of established infarcted regions, as determined by a late CT scan, had CMRO₂ values <1.3 mL · 100 g^-1 · min^-1 and that CMRO₂ was more predictive of tissue outcome than CBF, OEF, or CBV for the discrimination between viable and nonviable tissue. Ackerman et al,¹¹ in their study performed on 30 patients (5 within 2 to 6 hours), indicated that regions with CMRO₂ values <1.5 mL · 100 g^-1 · min^-1 always showed infarction on a late CT scan. Finally, Heiss and colleagues¹² reported that severely decreased CMRO₂ (by 55% on average [<1.3 mL · 100 g^-1 · min^-1] compared with its contralateral region) defines tissue that is without the capacity to regain functional recovery. In general, most authors suggest that a metabolic threshold >1.5 mL · 100 g^-1 · min^-1 is necessary to maintain structural integrity of the neuropil in humans. However, it should be noted that most of these studies (as a result of the complex logistics involved in PET technology) have been performed at least 6 hours after the ictus on patients who display a considerable clinical heterogeneity. Furthermore, only a few studies in a limited number of patients have followed regional metabolic alterations (with at best only two PET studies in each patient) to understand the transition from ischemia to infarction.^{23 33} In addition, since such analyses have often been performed on only one selected plane or at best on a summation of planes, no three-dimensional representation (ie, volume) of the ischemic and infarcted tissue could be quantified.

Anesthesia
In the present study PET examinations were performed under low-dose etomidate, which has been shown to be safe and to allow rapid recovery from anesthesia in both clinical and experimental investigations.^{13 34} A disadvantage of etomidate (with prolonged administration) is inhibition of adrenocortical steroidogenesis,²⁵ which may affect both blood volume and hematocrit. We counteracted this problem by the administration of concentrated erythrocytes at 7 hours after MCAO. In addition, clonidine potentiates the anesthetic effect and reduces the amount of anesthetics required.^{35 36} Greater bolus doses of etomidate, however, are known to lower CBF and CMRO₂ by approximately 43%,^{37 38 39} although this effect is transient.^{40 41} Our own PET studies were performed at least 5 hours after bolus injection of etomidate on the day of occlusion and at 2 hours for the other sequential studies (24 hours and the chronic examination).

Metabolic Thresholds in the Anesthetized Baboon
To characterize severely hypometabolic tissue and its temporal evolution, we used an objective PET data analysis method based on functional quantitative thresholds.^{22 23 42} This method allows one to follow every selected pixel for all PET measurements with respect to both physiological variables and time. As a first approach and in the absence of metabolic thresholds for irreversible damage in the nonhuman primate, we chose (based on the aforementioned clinical studies) an absolute value of 1.5 mL · 100 g^-1 · min^-1 (Table 2). This hypometabolic tissue volume increased with respect to time after occlusion. The hemispheric volume of hypometabolic tissue measured 17 days after MCAO was estimated to be 32% (13.1 cm³) of the contralateral hemispheric volume. This value was considerably greater than previously published infarct volumes obtained in a primate model of MCAO²⁶ and indeed considerably overestimated our own data obtained by histological analysis.

Based on these difficulties and on the fact that the metabolic threshold may not be constant throughout the time course of ischemia, we subsequently used a normalized (relative) threshold. Accordingly, we followed the evolution of tissue volume with a CMRO₂ <40%, <45%, and <50% of contralateral CMRO₂ (Table 3). By this procedure we could again clearly identify the extension of severely hypometabolic tissue volume as represented by these relative thresholds, and the profile of evolutivity was similar irrespective of the threshold used. More importantly, the final infarct volume as measured by quantitative histology was more consistent with the functional volume (measured in the chronic stage) with CMRO₂ <40% of contralateral CMRO₂ and correlated with tissue volumes with CMRO₂ <45% and <50%. Those PET-defined volumes might be affected by partial volume effect and overestimated by the inclusion of hypometabolic tissue caused by selective neuronal loss and/or disconnection in the infarct border,^{43 44} an effect perhaps enhanced by etomidate. However, whether peri-infarct neuronal loss exists in the primate brain remains controversial.^{45 46}

Since sequential PET examinations in this study were performed in healthy adolescent baboons in which MCAO produced only a small deep infarct, the assessment of the evolution of severely hypometabolic tissue volume may be more accurate in subjects with more extensively damaged regions. We show the feasibility of comparing a volume of infarction obtained by histological procedures and by using metabolic thresholds obtained by PET (even though the "infarct volume" identified by PET was based on only three 9-mm-thick planar sections, which may further affect the accuracy of measures²⁴ ). Because of the nonspecific and retarded histological changes (eg, brain shrinkage, ventricular dilatation), which would not be transposable on the metabolic images, we did not attempt here a voxel-by-voxel comparison of late histology with PET.

Evolution of Severe Hypometabolism
The data obtained in the present study demonstrate an extension of severely hypometabolic tissue volume—whether defined in absolute or relative CMRO₂ terms—after permanent MCAO. The volume of hypometabolic tissue was essentially stable at the 1-, 4-, and 7-hour postocclusion studies, increased markedly at the 24-hour study point, and increased even further in the chronic-stage study (on average, 17 days after occlusion). In the two baboons subjected to a repeated chronic study (one at 7 days and then at 22 days, the other at 14 days and then at 21 days after occlusion), the calculated volumes of severely hypometabolic tissue were essentially similar. Thus, the maximum infarct probably occurs later than 24 hours after MCAO. The difficulties inherent in repeated anesthesia precluded a more intensive study of the evolution of brain damage in the chronic stage, for which a different cohort of animals would need to be used, and it is clearly essential to define the evolution in the period after our 24-hour study.

This pattern of evolutivity observed in our study suggests that in the anesthetized baboon, the time to achieve a maximal irreversible lesion is extended for 24 hours after MCAO and perhaps even beyond. Based on a limited number of H₂ electrodes to measure CBF in a small tissue volume in the awake macaque monkey and a qualitative assessment of irreversibly damaged tissue, Jones et al²⁹ addressed the problem of the relationships between the duration of occlusion, the severity of ischemia, and the resulting tissue necrosis. These authors found microscopic foci of infarction after 15 to 30 minutes of ischemia, moderate to large infarcts after 2 to 3 hours of ischemia, and in most cases a large infarct after permanent MCAO. DeGirolami et al⁴ reported that consolidated necrosis was established after 8 hours of occlusion. However, it should be noted that these studies were strictly qualitative, and because of the methodology used, no evaluation of infarcted volume was attempted. In the study of Meier-Ruge and colleagues,⁴⁷ the time course of infarct volume was studied for 48 hours with an enzymatic staining technique in the macaque primate. This study (which closely parallels our functional investigation) indicated an extension of the nonviable focus as a function of time, with the infarct volume becoming maximal in the lenticulate nucleus at 24 hours and in the caudate nucleus at 48 hours after MCAO.

Conclusions
In our study the extension of severely hypometabolic tissue volume suggests the existence of potentially viable tissue in the border zone of ischemia, which gradually evolves toward irreversible damage. These penumbral border regions that evolve topographically with time are of great interest with regard to final outcome because they may represent brain tissue in which damage is still reversible and therefore amenable to therapeutic intervention.^{12 48} Therefore, serial multiparametric PET studies in the early and the chronic phases after insult might be of value in the assessment of characteristics of the penumbral zones and thus development of therapeutic strategies.

In the present study we attempted to analyze the pathophysiological characteristics of this tissue volume that will evolve toward a severely hypometabolic state in the chronic phase. At 1 hour this threatened tissue showed evidence of ischemia, with reduced CBF, increased OEF, and significantly decreased CMRO₂ compared with contralateral mirror tissue; this reduced CMRO₂ indicates neuronal dysfunction, as would be expected for a penumbral (at-risk) tissue. At 7 and 24 hours this surrounding tissue still showed hypoperfusion with worsening hypometabolism and a slight increase of OEF, which became mildly lower than the contralateral mirror tissue when analyzed at 24 hours. This profile of declining OEF as a result of deteriorating CMRO₂ has already been reported in previous clinical,^{12 33} baboon,⁶ and cat⁸ PET studies and marks the transition from penumbral ischemia to infarction.

	Selected Abbreviations and Acronyms

 

CBF = cerebral blood flow CBV = cerebral blood volume CMRO2 = cerebral metabolic rate of oxygen MCA = middle cerebral artery MCAO = middle cerebral artery occlusion OEF = oxygen extraction fraction PET = positron emission tomography

	Acknowledgments

 
This study was supported by the GIP Cyceron, the CEA, CNRS, INSERM, and by a grant from the Ministry of Research and Technology (89-C-0690). We are grateful to A. Brocquehaye and G. Huguet for technical assistance, the staff of the cyclotron unit, and the radiographers of the University Hospital of Caen.

Received January 11, 1995; revision received July 31, 1995; accepted August 1, 1995.

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