Exp Brain Res (1992) 89:67-78
Experimental BrainResearch 9 Springer-Verlag 1992
Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat H. Memezawa 1,2, H. Minamisawa 1,2, M-L. Smith 1, and B.K. Siesj6 ~ 1 Laboratory for Experimental Brain Research, Department of Neurobiology, Experimental Research Center, University Hospital, S-221 85 Lund, Sweden 2 Second Department of Internal Medicine, Nippon Medical School, Tokyo, Japan Received June 6, 1991 / Accepted December 3, 1991
Summary. It has become increasingly clear that a stroke lesion usually consists of a densely ischemic focus and of perifocal areas with better upheld flow rates. At least in rats and cats, some of these perifocal ("penumbral") areas subsequently become recruited in the infarction process. The mechanisms may involve an aberrant cellular calcium metabolism and enhanced production of free radicals. In general, though, the metabolic perturbation in the penumbra requires better characterization. The objective of this article was to define flow distribution in a rat model of reversible middle cerebral artery (MCA) occlusion, so as to allow delineation of the metabolic aberrations responsible for the subsequent infarction. We modified the intraluminal filament occlusion model recently developed by Koizumi et al. (1986), and described in more detail by Nagasawa and Kogure (1989), adopting it for use in both spontaneously breathing and artificially ventilated rats. Successful occlusion of the MCA (achieved in about 9/10 rats) was judged by unilateral EEG depression in ventilated rats, and neurological deficits, such as circling, in spontaneously breathing ones. CBF in the ipsilateral hemisphere was reduced to nearly constant values after 20, 60, and 120 rain of occlusion, flow rates in the focus being about 10% and in the perifocal ipsilateral areas about 15-20% of control (contralateral side). When the filament was left in place (permanent occlusion) 2,3,5-triphenyl tetrazolium chloride (TTC) staining and histopathology after 24 h showed a massive infarct on the occluded side, extending from caudoputamen and overlaying cortex to the occipital striate cortex. Animals recirculated after 60 rain of MCA occlusion, and allowed to survive 7 days for histopathology, showed infarction of the caudoputamen (lateral part or whole nucleus) in 5/6 animals and selective neuronal necrosis in one animal. The neocortex showed either infarcts, selective neuronal necrosis, or no damage. There was some overlap between neocortical areas which were infarcted and those which were salvaged by reperfusion. In general, though, both the CBF Offprint requests to: M-L. Smith
data and the recovery studies with a histopathological endpoint define large parts of the neocortex as perifocal (penumbral) areas which lend themselves to studies of metabolic events leading to infarction. Key words: Cerebral ischemia - Experimental stroke Recirculation - Cerebral blood flow - Brain damage Rat
Introduction In discussions of the pathophysiology of brain lesions caused by occlusion of the middle cerebral artery (MCA), the ischemic penumbra has received considerable attention. The term was originally coined to denote tissues which are sufficiently ischemic to lose their ability to generate spontaneous or evoked electrical activity, yet sufficiently nourished to maintain cellular membrane potentials and transmembrane ionic gradients (Astrup et al. 1981 ; Hakim 1987). The term can be used in a wider sense to denote those perifocal areas which are marginally supplied with oxygen, and which may be recruited in the infarction process unless normal perfusion is reestablished, or measures are instituted which prevent the cells from dying (for literature, see Nedergaard 1987; Siesj6 and Memezawa 1991 ; Symon 1980). This contention is based on the assumption that penumbral tissue is metabolically perturbed, and that the survival of its cells is jeopardized by events which gradually recruit damaged cells. These events appear to be related to an abnormal cellular calcium metabolism and to enhanced production of free radicals. The concept is supported by results showing that the treatment of rats and cats with N-methyl-D-aspartate (NMDA) antagonists or with some calcium antagonists reduces infarct size by about 50% (eg. Carter et al. 1988; Nakayama et al. 1988; Park et al. 1988a, b; Sauter and Rudin 1989; for overview, see Siesj6 et al. 1989), and that administration of free radical scavengers has a similar albeit somewhat less pronounced
68
effect (Liu et al. 1989; Martz et al. 1989). Such studies also suggest that a time window of 3-6 h exists before penumbral tissues are recruited in the infarction process. It is obviously of some importance to define perifocal events of putative pathogenetic importance. Important data have already been collected (Branston et al. 1974, 1977; Astrup et al. 1977; Strong et al. 1983; Nedergaard and Astrup 1986; Nedergaard et al. 1986), but much additional work is required before the pathophysiology of the ischemic lesion has been characterized. Progress in this field would be facilitated by the advent of stroke models, preferably in small animals like the rat, which allow reproducible induction of focal ischemia and clear identification of penumbral tissues. Ideally, the procedure should allow reperfusion to mimic recanalization of occluded vessels. If this is achieved penumbral tissues would be those salvaged if reperfusion is instituted before they have become overtly ischemic. One such model was described by Koizumi et al. 1986. These authors occluded the middle cerebral artery (MCA) by a silicon rubber coated thread, introduced via an incision in the common carotid artery. Since the thread can be pulled out, reversible ischemia is easily studied. The model was recently described in detail by Nagasawa and Kogure 1989 who determined intra- and postischemic cerebral blood flow rates. In this study, we used a slight modification of this embolic MCA occlusion model to characterize focal and penumbral events. We specially wished to identify the penumbra, i.e. those areas which initially have flow rates which are adequate for survival but which subsequently become recruited in the infarction process. To that end, we (a) determined regional flow rates after 20, 60, and 180 rain of ischemia, (b) assessed the size of the infarct after 24 h of permanent MCA occlusion, and (c) defined focal and perifocal structural alterations in animals surviving 7 days after 60 rain of reversible MCA occlusion. Since it took many months of
Table 1. Experimental subgroups
trial and error to achieve reproducible focal ischemia from the accounts published, we describe our own procedures, applicable to both spontaneously ventilating and artificially ventilated rats, in some detail. Material and methods Male Wistar rats (Mollegaard Breeding Center, Copenhagen, Denmark), weighing 290-350 g, were used for all experiments. The experimental procedures were approved by the Animal Use Ethical Committee of the University of Lund. The animals were fasted overnight before the day of the experiment, but allowed free access to tap water. They were divided into 12 experimental subgroups which are described in Table 1.
Preparation of experimental animals Anesthesia was induced with 3-3.5% halothane in N20/O 2 (70% :30%). All groups except 3 had their MCA occluded during artificial ventilation. The three exceptions were animals subjected to 20, 60, and 180 min ofischemia, with measurements of local CBF with the tissue sampling technique. After 60 rain, CBF was measured both by tissue sampling and by autoradiography (see below). The animals in the artificially ventilated groups were intubated and connected to a respirator, and the halothane concentration was reduced to 1-1.5%. The ventilation and oxygen admixture were adjusted to give an arterial PCO2 of 35-40 mmHg and a PO2 of close to or exceeding 100 mmHg. The animals in the spontaneously breathing groups continued to inhale 1.5-2% halothane. Polyethylene catheters were introduced into the tail artery for blood pressure recording and blood sampling, and a tail vein for drug/isotope infusions. In artificially ventilated animals used for measurements of CBF the femoral artery and vein were cannulated instead. EEG recordings were performed only in the artificially ventilated animals. For the right-hemispheric and conventional EEG recording, a pair of needle electrodes were inserted into the temporalis muscle bilaterally and a gold-plated screw was tapped on the exposed right frontal-parietal skull bone. A thermistor probe (type A-RM4, ELLAB A/S Copenhagen, Denmark) was placed on the bregma under the scalp to monitor and maintain the skull temperature at
Subgroups
Procedure
Ventilation
Numbers
AV AV AV
6 6 6
20 min MCAO 60 min MCAO 20 min MCAO 20 min MCAO 60 min MCAO 60 rain MCAO 180 min MCAO 60 min MCAO
AV AV SB AV SB AV SB SB
5 6 6 6 6 6 6 7
Permanent MCAO (28 days)
AV
1. Quantification of brain damage a. TTC staining 24 h MCAO b. Histopathology 24 h MCAO 60 min MCAO + 7 days reperfusion 2. Cerebral blood flow studies a. Carbon perfusion b. CBF: tissue sampling
c. CBF: autoradiography
3, Survival and behavior study
MCAO: middle cerebral artery occlusion AV: artificially ventilated, SB: spontaneously breathing
69 constant value (37 ~ C). The body temperature was also maintained at the same value by the use of the same type of thermistor probe in the rectum.
Operative proceduresfor middle cerebral artery occlusion (MCAO) The animal was placed in the supine position, and a skin incision was made in the midline of the neck. The right internal carotid artery was exposed from the carotid bifurcation to the basal cranium at the site of the tympanic bulla. The external carotid artery, which was also exposed, was then ligated by a 3-0 silk suture 1 mm distal from the carotid bifurcation. The pterygopalatine artery, which is a branch of the internal carotid artery, was encircled with a suture and retracted to the left side of the experimental animal to prevent incorrect insertion of the occluder. After these preparations, 0.1 ml of heparin (150 IU" ml -I) was given to the experimental animal. The MCA occluding device consisted of two pieces. The "occluder filament" was made from a nylon monofilament thread, 0.28 mm in diameter (sold as 0.25 ram, Platil, West Germany). Its tip was rounded and polished by no 1000 sandpaper with water, then cut to a length of 45 mm. It was inserted into a "guide sheath" made from a 30 mm long polyethylene catheter (PE-10, Portex Co, Kent, Occluder filament made from 0,280 nylon monofilament thread
Guide sheath made from polyethylene catileter j'
I
[]
(-- 10 "-) ( 30
9 1(-',
(
45ram
~'
~'
Fig. 1. Dimensions and appearance of the MCA occluding device. It consists of two pieces: occluder filament and guide sheath. The tip of the occluder filament (*) was rounded and polished by no 1000 sandpaper with water. Dimensions are in mm
A AcA
Mo?
England) which had a 2 mm long sleeve positioned 8 mm from the tip to work as a stopper (see Fig. 1). The internal carotid artery was occluded by a microvascular clip (Sugita-type, Mizuho Ikagaku Industries, Tokyo, Japan), and then the common carotid artery was closed by a suture, 3 mm proximal from the carotid bifurcation. A small incision was made in the common carotid artery 1 mm proximal from the carotid bifurcation, and the guide sheath with the MCA occluder was inserted into the internal carotid artery. After removal of the microvascular clip, the guide sheath was advanced 8 mm from the incision, passing through the pterygopalatine bifurcation. The guide sheath was secured by a suture, which had already occluded the common carotid artery, then the occluder filament inside the guide sheath was gently advanced 12 mm beyond the tip of the sheath. By this procedure, the tip of the occluder was lodged in the anterior cerebral artery, passing the MCA origin. The blood flow in the right MCA was blocked by the intraluminal occluder resulting in MCA occlusion (Fig. 2). In the rats used in the study of permanent MCA occlusion, the sutures which tied the guide sheath were tightened very carefully and the operative wound was closed.
Judgements of adequate MCA occlusion Adequate MCA occlusion was judged from the EEG in artificially ventilated animals, and from neurological behavior in spontaneously breathing ones. The EEG was used qualitatively to reveal whether a succesful occlusion had been achieved. As Fig. 4 shows, such occlusion caused severe suppression of the ipsilateral hemispheric record (B), as compared to the intrahemispheric record (A). Occasional animals with severe subarchnoidal hemhorrage (SAH, caused by a penetration of the internal carotid artery by the occluder filament) often showed suppression of intrahemispheric EEG as well. The neurological assessment was done by a modification of the method described by Bederson et al. (1986a). Spontaneously breathing animals with succesful MCA occlusion showed gait disturbances with circling or walking to the left. This neurological deficit is
EtA ,cA .."" carotidcanal
ccA Y f PPA
J I
B
C
Fig. 2A-C. Schematic diagram of anatomy of carotid arteries and explanation of the procedure used for MCA occlusion. A CCA; common carotid artery, ECA; external carotid artery, ICA; internal carotid artery, PPA; pterygopalatine artery, PCOM; posterior communicating artery, ACA; anterior cerebral artery, MCA; middle cerebral artery. B Insertion of MCA occluding device. The guide sheath was secured by a suture. C MCA occlusion performed by advancing the occluder filament
70 equal to the one described as grade 3 by Bederson et al. (1986a). In this study, rats showing grade. 1 neurological deficit (forelimb flexion), or grade 2 (decreased resistance to the lateral push) were regarded as having an unsatisfactory occlusion, in fact, these neurological deficits correlated to SAH. If a large SAH occured, the animal showed respiratory distress and often developed massive brain swelling. In animals which failed to show neurological deficits, the occluder filament did not obstruct the origin of the MCA. Only animals with circling or walking to the left were included in the study.
Brain tissue perfusion To define the distribution of the ischemic region, the brain was perfused with carbon black 20 and 60 rain after M C A occlusion. In 11 animals carbon black (Rotring Drawing ink suspension, Hamburg, West Germany) was perfused via the ascending aorta after flushing with saline. The brains were immersed in phosphate buffered 4% formaldehyde (pH 7.4) for 1 week and were cut in the coronal plane at the levels of caudoputamen (CPu), hippocampus, and substantia nigra (SN). Thirty-seven animals were used to study CBF. Thirty animals were divided into 5 groups of 6 animals each. These were used for regional CBF studies by the tissue sampling technique described by Ekl/Sf and Siesj8 1973. The animals were studied after 20 and 60 rain of ischemia under artificial ventilation, and after 20, 60 and 180 min of ischemia under spontaneous breathing (see Table 1, above). ~4C-iodoantipyrine was used as the diffusible tracer, with 15 gCi of the isotope being infused at constant rate during 45 s. In the spontaneously breathing groups, the animals were allowed to wake up to confirm the presence of neurological deficits. They were thereafter placed in a small plexi-glass cage, restricting the movements of the animal, and giving access to the catheters for blood sampling and infusions. Repeated, timed blood samples for [3-scintillation counting of :4C-activity were taken from the femoral artery during the tracer infusion. Simultaneously with the last blood sample the animal was decapitated. The brain samples were dissected from coronal sections of 1.5 mm thickness through the caudoputamen, and they were cut into 5 pieces from each hemispheres according to Fig. 3. The samples were weighed and put into plastic vials containing Soluene| and allowed to stand in the dark for 24 h to be dissolved. The :4C activities of brain samples and the blood were counted in a liquid scintillation counter. The CBF was calculated as described by Sakurada et al. 1978. The statistical signifi-
cance between all groups was evaluated by Scheffe's F-test in each region. In the remaining 7 animals, subjected to 60 rain of M C A occlusion, local CBF was measured with the autoradiographic technique of Sakurada et al. (35), as described by Abdul-Rahman et al. 1980 and Ingvar and Siesj8 1983. ~4C-iodoantipyrine was used as the diffusible tracer. The isotope (30 gCi) was infused i.v. at a constant rate for 60 s. Blood sampling and decapitation were performed as in the tissue sampling-CBF study. The brains were sectioned coronally at 20 pm in a cryostat at - 2 0 ~ C and exposed to an X-ray film for one week together with a set of calibrated :4C standards. After development of the exposed film the brain structures were evaluated with the help of an IBAS 2 image analyzer (Kontron Bildanalyse GMBH, Munich, West Germany) with a geometrical resolution of 512x 512 pixels and a gray value resolution of 256 steps. The autoradiographic image of the brain sections were magnified so that after digitalization, 25 pixels on the digital image corresponded to 1 mm on the original autoradiographic film. The brain structures examined were determined according to the stereotaxic rat brain atlas of Paxinos and Watson 1982. The gray value for each interactively selected brain structure was measured bilaterally on 3 dif-
before MCA occlusion " _k
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Fig. 3. Anatomic regions of neocortex and caudoputamen used for measurement of regional CBF by tissue sampling technique. Five pieces of 1.5 mm thickness were cut out from each hemisphere as illustrated. CX-1 ; frontoparietal cortex motor area, CX-2: upper part of frontoparietal somatosensory area, CX-3: lower part of frontoparietal somatosensory area, CP-m: medial caudoputamen, and CP-1 : lateral caudoputamen
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Fig. 4A, B. Differences on EEG recording before and during MCA occlusion. Bipolar EEG was recorded by 2 ways; A: between both hemispheres, and B: right hemisphere. M C A occlusion produced a 9marked depression of the right hemispheric EEG
71 ferent sections and the mean values for each hemisphere were used for calculations of 1-CBF as described by Sakurada et al. 1978. Statistical differences between the occluded and contralateral hemisphere were evaluated by Scheffe's F-test.
Quantification of brain damage after permanent ischemia and long~term reperfusion In these groups, operations were performed under artificial ventilation. After occlusion of the MCA the animals were allowed to recover from anesthesia and were disconnected from the respirator. During the recovery period, they had free access to food pellets and water. After 24 h of MCA occlusion six animals were reanesthetized with 3-3.5% halothane and following decapitation, their brains were removed. The brains, which were cut in the same way as in the carbon perfused group, were immersed in 2% 2,3,5-triphenyl tetrazolium chloride (TTC) for 30 min (Liszczak et al. 1984, as modified by Bederson et al. 1986b). We assessed these brains assuming that infarcted areas lacked staining with TTC. Six other animals were used for histopathological evaluation. They were reanesthetized after 24 h, tracheotomized and artificially ventilated with 1.0 % halothane in NzO/O2 (70 % : 30 %). A thoracotomy was made and a cannula was inserted into the ascending aorta via the left ventricle. After a short flush with saline, the animals were perfusion-fixed with phosphate buffered 4 % formaldehyde (pH 7.4). Then the brains were removed from the cranium, dehydrated, embedded in paraffin, sectioned coronally at 8 pm and stained with a combination of celestine blue and acid fuchsin (Auer et al. 1985). Six animals were reperfused after 60 min of MCA occlusion. The sutures which secured the MCA occluder were loosened and the MCA occluder was removed together with the guide sheath. The sutures were immediately tightened to prevent bleeding. The wound was then closed and the animals were extubated and placed in cages with free access to pellets and water. They were perfusion-fixed 7 days later and histopathologically examined. Quantification of the infarcted area was performed on three brain levels (caudoputamen, hippocampus, and substantia nigra). The areas of the infarct and the occluded hemisphere were measured with an IBAS 2 image analyzer (see above) and the percentage of infarcted tissue was calculated. Statistical differences between the TTC stained group and the histopathologically examined group with 24 h ischemia, and between 24 h ischemia and 60 min of ischemia followed by 7 days reperfusion in histopathologically examined groups were evaluated by Scheffe's F-test.
Long term survival and behaviour Four animals were observed during permanent MCA occlusion. They had free access to pellets and water. The body weight and neurological behavior of the experimental animals were checked. The two animals which survived for 4 weeks were perfusion-fixed.
Results
T h e a v e r a g e o p e r a t i o n time for M C A o c c l u s i o n ( f r o m the n e c k incision to M C A o c c l u d e r i n s e r t i o n ) w a s between 20 a n d 30 rnin. Difficulties were sometimes e n c o u n t e r e d b y k i n k s in the i n t e r n a l c a r o t i d artery. I n such cases, the difficulties c o u l d u s u a l l y be a v o i d e d b y p u l l i n g the a r t e r y o v e r the s h e a t h w h i c h h a d a l r e a d y been positioned. A n i m a l s w h i c h r e q u i r e d a n o p e r a t i o n time exceeding 30 m i n were o m i t t e d f r o m the m a t e r i a l . I n the series o f e x p e r i m e n t s r e p o r t e d here, o p e r a t i o n failure c a u s e d b y p e n e t r a t i o n o f the i n t e r n a l c a r o t i d artery was a b o u t 10%. T h e p r o p o r t i o n o f r a t s w h i c h failed to s h o w the e x p e c t e d c h a n g e s in E E G a n d n e u r o g i c a l b e h a v i o r was 17 %. W i t h t i m e the t e c h n i q u e for i n s e r t i o n o f the o c c l u d e r filament h a s been i m p r o v e d , a n d r e c e n t l y the p e r c e n t a g e o f successful e x p e r i m e n t s is a r o u n d 90 %.
Physiological variables T a b l e 2 shows p h y s i o l o g i c a l p a r a m e t e r s , as m e a s u r e d 15-20 m i n after M C A o c c l u s i o n (20 a n d 60 m i n occlusion), o r 170-175 m i n after M C A o c c l u s i o n ( s p o n t a n e o u s l y b r e a t h i n g a n i m a l s w i t h 180 rain occlusion). B l o o d p r e s s u r e , PO2, p H , a n d glucose c o n c e n t r a t i o n were similar in all g r o u p s , b u t P C O 2 was initially inc r e a s e d in s p o n t a n e o u s l y b r e a t h i n g a n i m a l s ( p < 0 . 0 1 ) .
Changes of focal and perifocaI blood flow during MCA occlusion P e r f u s i o n w i t h c a r b o n b l a c k was used to o b t a i n a q u a n tative e s t i m a t e o f the a r e a o f u n d e r p e r f u s i o n . A f t e r 20 rain, p o o r filling o f vessels with c a r b o n b l a c k was f o u n d in the i p s i l a t e r a l h e m i s p h e r e , affecting the M C A d i s t r i b u t i o n t e r r i t o r y . T h e p e r f u s i o n deficit was s o m e w h a t variable. I n o n e a n i m a l , o n l y the l a t e r a l p a r t o f the c a u d o p u t a m e n a n d d e e p e r layers o f the o v e r l a y i n g c o r t e x were affected. I n all others, the defect affected v i r t u a l l y the w h o l e c a u d o p u t a m e n a n d v a r i a b l e a m o u n t s o f n e o cortex. A f t e r 60 min, the p e r f u s i o n defects were m o r e u n i f o r m (Fig. 5). O n the basis o f these results, s a m p l i n g o f tissue for C B F m e a s u r e m e n t s w a s p e r f o r m e d as des c r i b e d a b o v e (see Fig. 3 above).
Table 2. Physiological parameters in the regional CBF study (tissue sampling)
BP PCO 2 PO 2 pH Blood glucose
(mmHg) (mmHg) (mmHg) (mmol.1-1)
SB-20
AV-20
SB-60
AV-60
SB-180"
123 :t:5 55.1 • b,~ 125 + 9.0 7.32 4- 0.18 c 5.5 4-0.1
115 4-4 39.5 4-1.2 106 4- 5.0 7.38 • 0.02 5.4 4-0.1
118 4-5 47.9 • b 128 4- 3.7 7.39 4- 0.16 5.5 4-0.2
127 4-6 40.1 • 107 4- 3.0 7.39 4- 0.01 5.9 4-0.3
131 • 5 35.8 • 1.9 134 + 16 7.42 4- 0.01 6.5 4- 0.4
Values are mean 4-S.E., n = 6 in each group SB: spontaneously breathing, AV: artificially ventilated Blood samples were taken 15-20 min (" 170-175 min) after introduction of MCA occlusion
0.01 compared to corresponding AV group by Scheffe's F-test ~p < 0.01 compared to SB-180 group by Scheffe's F-test
bp
Experimental BrainResearch 9 Springer-Verlag 1992
Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat H. Memezawa 1,2, H. Minamisawa 1,2, M-L. Smith 1, and B.K. Siesj6 ~ 1 Laboratory for Experimental Brain Research, Department of Neurobiology, Experimental Research Center, University Hospital, S-221 85 Lund, Sweden 2 Second Department of Internal Medicine, Nippon Medical School, Tokyo, Japan Received June 6, 1991 / Accepted December 3, 1991
Summary. It has become increasingly clear that a stroke lesion usually consists of a densely ischemic focus and of perifocal areas with better upheld flow rates. At least in rats and cats, some of these perifocal ("penumbral") areas subsequently become recruited in the infarction process. The mechanisms may involve an aberrant cellular calcium metabolism and enhanced production of free radicals. In general, though, the metabolic perturbation in the penumbra requires better characterization. The objective of this article was to define flow distribution in a rat model of reversible middle cerebral artery (MCA) occlusion, so as to allow delineation of the metabolic aberrations responsible for the subsequent infarction. We modified the intraluminal filament occlusion model recently developed by Koizumi et al. (1986), and described in more detail by Nagasawa and Kogure (1989), adopting it for use in both spontaneously breathing and artificially ventilated rats. Successful occlusion of the MCA (achieved in about 9/10 rats) was judged by unilateral EEG depression in ventilated rats, and neurological deficits, such as circling, in spontaneously breathing ones. CBF in the ipsilateral hemisphere was reduced to nearly constant values after 20, 60, and 120 rain of occlusion, flow rates in the focus being about 10% and in the perifocal ipsilateral areas about 15-20% of control (contralateral side). When the filament was left in place (permanent occlusion) 2,3,5-triphenyl tetrazolium chloride (TTC) staining and histopathology after 24 h showed a massive infarct on the occluded side, extending from caudoputamen and overlaying cortex to the occipital striate cortex. Animals recirculated after 60 rain of MCA occlusion, and allowed to survive 7 days for histopathology, showed infarction of the caudoputamen (lateral part or whole nucleus) in 5/6 animals and selective neuronal necrosis in one animal. The neocortex showed either infarcts, selective neuronal necrosis, or no damage. There was some overlap between neocortical areas which were infarcted and those which were salvaged by reperfusion. In general, though, both the CBF Offprint requests to: M-L. Smith
data and the recovery studies with a histopathological endpoint define large parts of the neocortex as perifocal (penumbral) areas which lend themselves to studies of metabolic events leading to infarction. Key words: Cerebral ischemia - Experimental stroke Recirculation - Cerebral blood flow - Brain damage Rat
Introduction In discussions of the pathophysiology of brain lesions caused by occlusion of the middle cerebral artery (MCA), the ischemic penumbra has received considerable attention. The term was originally coined to denote tissues which are sufficiently ischemic to lose their ability to generate spontaneous or evoked electrical activity, yet sufficiently nourished to maintain cellular membrane potentials and transmembrane ionic gradients (Astrup et al. 1981 ; Hakim 1987). The term can be used in a wider sense to denote those perifocal areas which are marginally supplied with oxygen, and which may be recruited in the infarction process unless normal perfusion is reestablished, or measures are instituted which prevent the cells from dying (for literature, see Nedergaard 1987; Siesj6 and Memezawa 1991 ; Symon 1980). This contention is based on the assumption that penumbral tissue is metabolically perturbed, and that the survival of its cells is jeopardized by events which gradually recruit damaged cells. These events appear to be related to an abnormal cellular calcium metabolism and to enhanced production of free radicals. The concept is supported by results showing that the treatment of rats and cats with N-methyl-D-aspartate (NMDA) antagonists or with some calcium antagonists reduces infarct size by about 50% (eg. Carter et al. 1988; Nakayama et al. 1988; Park et al. 1988a, b; Sauter and Rudin 1989; for overview, see Siesj6 et al. 1989), and that administration of free radical scavengers has a similar albeit somewhat less pronounced
68
effect (Liu et al. 1989; Martz et al. 1989). Such studies also suggest that a time window of 3-6 h exists before penumbral tissues are recruited in the infarction process. It is obviously of some importance to define perifocal events of putative pathogenetic importance. Important data have already been collected (Branston et al. 1974, 1977; Astrup et al. 1977; Strong et al. 1983; Nedergaard and Astrup 1986; Nedergaard et al. 1986), but much additional work is required before the pathophysiology of the ischemic lesion has been characterized. Progress in this field would be facilitated by the advent of stroke models, preferably in small animals like the rat, which allow reproducible induction of focal ischemia and clear identification of penumbral tissues. Ideally, the procedure should allow reperfusion to mimic recanalization of occluded vessels. If this is achieved penumbral tissues would be those salvaged if reperfusion is instituted before they have become overtly ischemic. One such model was described by Koizumi et al. 1986. These authors occluded the middle cerebral artery (MCA) by a silicon rubber coated thread, introduced via an incision in the common carotid artery. Since the thread can be pulled out, reversible ischemia is easily studied. The model was recently described in detail by Nagasawa and Kogure 1989 who determined intra- and postischemic cerebral blood flow rates. In this study, we used a slight modification of this embolic MCA occlusion model to characterize focal and penumbral events. We specially wished to identify the penumbra, i.e. those areas which initially have flow rates which are adequate for survival but which subsequently become recruited in the infarction process. To that end, we (a) determined regional flow rates after 20, 60, and 180 rain of ischemia, (b) assessed the size of the infarct after 24 h of permanent MCA occlusion, and (c) defined focal and perifocal structural alterations in animals surviving 7 days after 60 rain of reversible MCA occlusion. Since it took many months of
Table 1. Experimental subgroups
trial and error to achieve reproducible focal ischemia from the accounts published, we describe our own procedures, applicable to both spontaneously ventilating and artificially ventilated rats, in some detail. Material and methods Male Wistar rats (Mollegaard Breeding Center, Copenhagen, Denmark), weighing 290-350 g, were used for all experiments. The experimental procedures were approved by the Animal Use Ethical Committee of the University of Lund. The animals were fasted overnight before the day of the experiment, but allowed free access to tap water. They were divided into 12 experimental subgroups which are described in Table 1.
Preparation of experimental animals Anesthesia was induced with 3-3.5% halothane in N20/O 2 (70% :30%). All groups except 3 had their MCA occluded during artificial ventilation. The three exceptions were animals subjected to 20, 60, and 180 min ofischemia, with measurements of local CBF with the tissue sampling technique. After 60 rain, CBF was measured both by tissue sampling and by autoradiography (see below). The animals in the artificially ventilated groups were intubated and connected to a respirator, and the halothane concentration was reduced to 1-1.5%. The ventilation and oxygen admixture were adjusted to give an arterial PCO2 of 35-40 mmHg and a PO2 of close to or exceeding 100 mmHg. The animals in the spontaneously breathing groups continued to inhale 1.5-2% halothane. Polyethylene catheters were introduced into the tail artery for blood pressure recording and blood sampling, and a tail vein for drug/isotope infusions. In artificially ventilated animals used for measurements of CBF the femoral artery and vein were cannulated instead. EEG recordings were performed only in the artificially ventilated animals. For the right-hemispheric and conventional EEG recording, a pair of needle electrodes were inserted into the temporalis muscle bilaterally and a gold-plated screw was tapped on the exposed right frontal-parietal skull bone. A thermistor probe (type A-RM4, ELLAB A/S Copenhagen, Denmark) was placed on the bregma under the scalp to monitor and maintain the skull temperature at
Subgroups
Procedure
Ventilation
Numbers
AV AV AV
6 6 6
20 min MCAO 60 min MCAO 20 min MCAO 20 min MCAO 60 min MCAO 60 rain MCAO 180 min MCAO 60 min MCAO
AV AV SB AV SB AV SB SB
5 6 6 6 6 6 6 7
Permanent MCAO (28 days)
AV
1. Quantification of brain damage a. TTC staining 24 h MCAO b. Histopathology 24 h MCAO 60 min MCAO + 7 days reperfusion 2. Cerebral blood flow studies a. Carbon perfusion b. CBF: tissue sampling
c. CBF: autoradiography
3, Survival and behavior study
MCAO: middle cerebral artery occlusion AV: artificially ventilated, SB: spontaneously breathing
69 constant value (37 ~ C). The body temperature was also maintained at the same value by the use of the same type of thermistor probe in the rectum.
Operative proceduresfor middle cerebral artery occlusion (MCAO) The animal was placed in the supine position, and a skin incision was made in the midline of the neck. The right internal carotid artery was exposed from the carotid bifurcation to the basal cranium at the site of the tympanic bulla. The external carotid artery, which was also exposed, was then ligated by a 3-0 silk suture 1 mm distal from the carotid bifurcation. The pterygopalatine artery, which is a branch of the internal carotid artery, was encircled with a suture and retracted to the left side of the experimental animal to prevent incorrect insertion of the occluder. After these preparations, 0.1 ml of heparin (150 IU" ml -I) was given to the experimental animal. The MCA occluding device consisted of two pieces. The "occluder filament" was made from a nylon monofilament thread, 0.28 mm in diameter (sold as 0.25 ram, Platil, West Germany). Its tip was rounded and polished by no 1000 sandpaper with water, then cut to a length of 45 mm. It was inserted into a "guide sheath" made from a 30 mm long polyethylene catheter (PE-10, Portex Co, Kent, Occluder filament made from 0,280 nylon monofilament thread
Guide sheath made from polyethylene catileter j'
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Fig. 1. Dimensions and appearance of the MCA occluding device. It consists of two pieces: occluder filament and guide sheath. The tip of the occluder filament (*) was rounded and polished by no 1000 sandpaper with water. Dimensions are in mm
A AcA
Mo?
England) which had a 2 mm long sleeve positioned 8 mm from the tip to work as a stopper (see Fig. 1). The internal carotid artery was occluded by a microvascular clip (Sugita-type, Mizuho Ikagaku Industries, Tokyo, Japan), and then the common carotid artery was closed by a suture, 3 mm proximal from the carotid bifurcation. A small incision was made in the common carotid artery 1 mm proximal from the carotid bifurcation, and the guide sheath with the MCA occluder was inserted into the internal carotid artery. After removal of the microvascular clip, the guide sheath was advanced 8 mm from the incision, passing through the pterygopalatine bifurcation. The guide sheath was secured by a suture, which had already occluded the common carotid artery, then the occluder filament inside the guide sheath was gently advanced 12 mm beyond the tip of the sheath. By this procedure, the tip of the occluder was lodged in the anterior cerebral artery, passing the MCA origin. The blood flow in the right MCA was blocked by the intraluminal occluder resulting in MCA occlusion (Fig. 2). In the rats used in the study of permanent MCA occlusion, the sutures which tied the guide sheath were tightened very carefully and the operative wound was closed.
Judgements of adequate MCA occlusion Adequate MCA occlusion was judged from the EEG in artificially ventilated animals, and from neurological behavior in spontaneously breathing ones. The EEG was used qualitatively to reveal whether a succesful occlusion had been achieved. As Fig. 4 shows, such occlusion caused severe suppression of the ipsilateral hemispheric record (B), as compared to the intrahemispheric record (A). Occasional animals with severe subarchnoidal hemhorrage (SAH, caused by a penetration of the internal carotid artery by the occluder filament) often showed suppression of intrahemispheric EEG as well. The neurological assessment was done by a modification of the method described by Bederson et al. (1986a). Spontaneously breathing animals with succesful MCA occlusion showed gait disturbances with circling or walking to the left. This neurological deficit is
EtA ,cA .."" carotidcanal
ccA Y f PPA
J I
B
C
Fig. 2A-C. Schematic diagram of anatomy of carotid arteries and explanation of the procedure used for MCA occlusion. A CCA; common carotid artery, ECA; external carotid artery, ICA; internal carotid artery, PPA; pterygopalatine artery, PCOM; posterior communicating artery, ACA; anterior cerebral artery, MCA; middle cerebral artery. B Insertion of MCA occluding device. The guide sheath was secured by a suture. C MCA occlusion performed by advancing the occluder filament
70 equal to the one described as grade 3 by Bederson et al. (1986a). In this study, rats showing grade. 1 neurological deficit (forelimb flexion), or grade 2 (decreased resistance to the lateral push) were regarded as having an unsatisfactory occlusion, in fact, these neurological deficits correlated to SAH. If a large SAH occured, the animal showed respiratory distress and often developed massive brain swelling. In animals which failed to show neurological deficits, the occluder filament did not obstruct the origin of the MCA. Only animals with circling or walking to the left were included in the study.
Brain tissue perfusion To define the distribution of the ischemic region, the brain was perfused with carbon black 20 and 60 rain after M C A occlusion. In 11 animals carbon black (Rotring Drawing ink suspension, Hamburg, West Germany) was perfused via the ascending aorta after flushing with saline. The brains were immersed in phosphate buffered 4% formaldehyde (pH 7.4) for 1 week and were cut in the coronal plane at the levels of caudoputamen (CPu), hippocampus, and substantia nigra (SN). Thirty-seven animals were used to study CBF. Thirty animals were divided into 5 groups of 6 animals each. These were used for regional CBF studies by the tissue sampling technique described by Ekl/Sf and Siesj8 1973. The animals were studied after 20 and 60 rain of ischemia under artificial ventilation, and after 20, 60 and 180 min of ischemia under spontaneous breathing (see Table 1, above). ~4C-iodoantipyrine was used as the diffusible tracer, with 15 gCi of the isotope being infused at constant rate during 45 s. In the spontaneously breathing groups, the animals were allowed to wake up to confirm the presence of neurological deficits. They were thereafter placed in a small plexi-glass cage, restricting the movements of the animal, and giving access to the catheters for blood sampling and infusions. Repeated, timed blood samples for [3-scintillation counting of :4C-activity were taken from the femoral artery during the tracer infusion. Simultaneously with the last blood sample the animal was decapitated. The brain samples were dissected from coronal sections of 1.5 mm thickness through the caudoputamen, and they were cut into 5 pieces from each hemispheres according to Fig. 3. The samples were weighed and put into plastic vials containing Soluene| and allowed to stand in the dark for 24 h to be dissolved. The :4C activities of brain samples and the blood were counted in a liquid scintillation counter. The CBF was calculated as described by Sakurada et al. 1978. The statistical signifi-
cance between all groups was evaluated by Scheffe's F-test in each region. In the remaining 7 animals, subjected to 60 rain of M C A occlusion, local CBF was measured with the autoradiographic technique of Sakurada et al. (35), as described by Abdul-Rahman et al. 1980 and Ingvar and Siesj8 1983. ~4C-iodoantipyrine was used as the diffusible tracer. The isotope (30 gCi) was infused i.v. at a constant rate for 60 s. Blood sampling and decapitation were performed as in the tissue sampling-CBF study. The brains were sectioned coronally at 20 pm in a cryostat at - 2 0 ~ C and exposed to an X-ray film for one week together with a set of calibrated :4C standards. After development of the exposed film the brain structures were evaluated with the help of an IBAS 2 image analyzer (Kontron Bildanalyse GMBH, Munich, West Germany) with a geometrical resolution of 512x 512 pixels and a gray value resolution of 256 steps. The autoradiographic image of the brain sections were magnified so that after digitalization, 25 pixels on the digital image corresponded to 1 mm on the original autoradiographic film. The brain structures examined were determined according to the stereotaxic rat brain atlas of Paxinos and Watson 1982. The gray value for each interactively selected brain structure was measured bilaterally on 3 dif-
before MCA occlusion " _k
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Fig. 3. Anatomic regions of neocortex and caudoputamen used for measurement of regional CBF by tissue sampling technique. Five pieces of 1.5 mm thickness were cut out from each hemisphere as illustrated. CX-1 ; frontoparietal cortex motor area, CX-2: upper part of frontoparietal somatosensory area, CX-3: lower part of frontoparietal somatosensory area, CP-m: medial caudoputamen, and CP-1 : lateral caudoputamen
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Fig. 4A, B. Differences on EEG recording before and during MCA occlusion. Bipolar EEG was recorded by 2 ways; A: between both hemispheres, and B: right hemisphere. M C A occlusion produced a 9marked depression of the right hemispheric EEG
71 ferent sections and the mean values for each hemisphere were used for calculations of 1-CBF as described by Sakurada et al. 1978. Statistical differences between the occluded and contralateral hemisphere were evaluated by Scheffe's F-test.
Quantification of brain damage after permanent ischemia and long~term reperfusion In these groups, operations were performed under artificial ventilation. After occlusion of the MCA the animals were allowed to recover from anesthesia and were disconnected from the respirator. During the recovery period, they had free access to food pellets and water. After 24 h of MCA occlusion six animals were reanesthetized with 3-3.5% halothane and following decapitation, their brains were removed. The brains, which were cut in the same way as in the carbon perfused group, were immersed in 2% 2,3,5-triphenyl tetrazolium chloride (TTC) for 30 min (Liszczak et al. 1984, as modified by Bederson et al. 1986b). We assessed these brains assuming that infarcted areas lacked staining with TTC. Six other animals were used for histopathological evaluation. They were reanesthetized after 24 h, tracheotomized and artificially ventilated with 1.0 % halothane in NzO/O2 (70 % : 30 %). A thoracotomy was made and a cannula was inserted into the ascending aorta via the left ventricle. After a short flush with saline, the animals were perfusion-fixed with phosphate buffered 4 % formaldehyde (pH 7.4). Then the brains were removed from the cranium, dehydrated, embedded in paraffin, sectioned coronally at 8 pm and stained with a combination of celestine blue and acid fuchsin (Auer et al. 1985). Six animals were reperfused after 60 min of MCA occlusion. The sutures which secured the MCA occluder were loosened and the MCA occluder was removed together with the guide sheath. The sutures were immediately tightened to prevent bleeding. The wound was then closed and the animals were extubated and placed in cages with free access to pellets and water. They were perfusion-fixed 7 days later and histopathologically examined. Quantification of the infarcted area was performed on three brain levels (caudoputamen, hippocampus, and substantia nigra). The areas of the infarct and the occluded hemisphere were measured with an IBAS 2 image analyzer (see above) and the percentage of infarcted tissue was calculated. Statistical differences between the TTC stained group and the histopathologically examined group with 24 h ischemia, and between 24 h ischemia and 60 min of ischemia followed by 7 days reperfusion in histopathologically examined groups were evaluated by Scheffe's F-test.
Long term survival and behaviour Four animals were observed during permanent MCA occlusion. They had free access to pellets and water. The body weight and neurological behavior of the experimental animals were checked. The two animals which survived for 4 weeks were perfusion-fixed.
Results
T h e a v e r a g e o p e r a t i o n time for M C A o c c l u s i o n ( f r o m the n e c k incision to M C A o c c l u d e r i n s e r t i o n ) w a s between 20 a n d 30 rnin. Difficulties were sometimes e n c o u n t e r e d b y k i n k s in the i n t e r n a l c a r o t i d artery. I n such cases, the difficulties c o u l d u s u a l l y be a v o i d e d b y p u l l i n g the a r t e r y o v e r the s h e a t h w h i c h h a d a l r e a d y been positioned. A n i m a l s w h i c h r e q u i r e d a n o p e r a t i o n time exceeding 30 m i n were o m i t t e d f r o m the m a t e r i a l . I n the series o f e x p e r i m e n t s r e p o r t e d here, o p e r a t i o n failure c a u s e d b y p e n e t r a t i o n o f the i n t e r n a l c a r o t i d artery was a b o u t 10%. T h e p r o p o r t i o n o f r a t s w h i c h failed to s h o w the e x p e c t e d c h a n g e s in E E G a n d n e u r o g i c a l b e h a v i o r was 17 %. W i t h t i m e the t e c h n i q u e for i n s e r t i o n o f the o c c l u d e r filament h a s been i m p r o v e d , a n d r e c e n t l y the p e r c e n t a g e o f successful e x p e r i m e n t s is a r o u n d 90 %.
Physiological variables T a b l e 2 shows p h y s i o l o g i c a l p a r a m e t e r s , as m e a s u r e d 15-20 m i n after M C A o c c l u s i o n (20 a n d 60 m i n occlusion), o r 170-175 m i n after M C A o c c l u s i o n ( s p o n t a n e o u s l y b r e a t h i n g a n i m a l s w i t h 180 rain occlusion). B l o o d p r e s s u r e , PO2, p H , a n d glucose c o n c e n t r a t i o n were similar in all g r o u p s , b u t P C O 2 was initially inc r e a s e d in s p o n t a n e o u s l y b r e a t h i n g a n i m a l s ( p < 0 . 0 1 ) .
Changes of focal and perifocaI blood flow during MCA occlusion P e r f u s i o n w i t h c a r b o n b l a c k was used to o b t a i n a q u a n tative e s t i m a t e o f the a r e a o f u n d e r p e r f u s i o n . A f t e r 20 rain, p o o r filling o f vessels with c a r b o n b l a c k was f o u n d in the i p s i l a t e r a l h e m i s p h e r e , affecting the M C A d i s t r i b u t i o n t e r r i t o r y . T h e p e r f u s i o n deficit was s o m e w h a t variable. I n o n e a n i m a l , o n l y the l a t e r a l p a r t o f the c a u d o p u t a m e n a n d d e e p e r layers o f the o v e r l a y i n g c o r t e x were affected. I n all others, the defect affected v i r t u a l l y the w h o l e c a u d o p u t a m e n a n d v a r i a b l e a m o u n t s o f n e o cortex. A f t e r 60 min, the p e r f u s i o n defects were m o r e u n i f o r m (Fig. 5). O n the basis o f these results, s a m p l i n g o f tissue for C B F m e a s u r e m e n t s w a s p e r f o r m e d as des c r i b e d a b o v e (see Fig. 3 above).
Table 2. Physiological parameters in the regional CBF study (tissue sampling)
BP PCO 2 PO 2 pH Blood glucose
(mmHg) (mmHg) (mmHg) (mmol.1-1)
SB-20
AV-20
SB-60
AV-60
SB-180"
123 :t:5 55.1 • b,~ 125 + 9.0 7.32 4- 0.18 c 5.5 4-0.1
115 4-4 39.5 4-1.2 106 4- 5.0 7.38 • 0.02 5.4 4-0.1
118 4-5 47.9 • b 128 4- 3.7 7.39 4- 0.16 5.5 4-0.2
127 4-6 40.1 • 107 4- 3.0 7.39 4- 0.01 5.9 4-0.3
131 • 5 35.8 • 1.9 134 + 16 7.42 4- 0.01 6.5 4- 0.4
Values are mean 4-S.E., n = 6 in each group SB: spontaneously breathing, AV: artificially ventilated Blood samples were taken 15-20 min (" 170-175 min) after introduction of MCA occlusion
0.01 compared to corresponding AV group by Scheffe's F-test ~p < 0.01 compared to SB-180 group by Scheffe's F-test
bp
