Neuroscienceand BiobehavioralReviews, Vol. 16, pp. 219-233, 1992
0149-7634/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
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Genetic Hypertension and Increased Susceptibility to Cerebral Ischemia 1 F.
C.
BARONE,
W.
J.
PRICE,
R.
F.
WHITE,
R.
N.
WlLLETTE
AND
G.
Z.
FEUERSTEIN
D e p a r t m e n t o f P h a r m a c o l o g y , S m i t h K l i n e B e e c h a m Pharmaceuticals, plc, K i n g o f Prussia, P A 19406 Received 5 August
1991
B A R O N E , F. C., W. J. PRICE, R. F. W H I T E , R. N. W I L L E T T E A N D G. Z. FEUERSTEIN. Genetic hypertension and increased susceptibility to cerebral ischemia. NEUROSCI BIOBEHAV REV 16(2) 219-233, 1 9 9 2 . - A review of the sensitivity of genetically hypertensive rats to cerebral ischemia was presented together with original data describing the systematic comparison of the effects of focal ischemia (permanent and temporary with reperfusion) performed in hypertensive and normotensive rats (i.e., blood pressures verified in conscious instrumented rats). Microsurgical techniques were used to isolate and occlude the middle cerebral artery (MCAO) of spontaneously hypertensive (SHR), Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats at the level of the inferior cerebral vein. Following permanent (24 h) M C A O , persistent and similar decreases in local microvascular perfusion (i.e., to 15.6 _+ 1.7070 of pre-MCAO levels) were verified in the primary ischemic zone of the cortex for all strains using Laser-Doppler flowmetry. A contralateral hemiplegia that occurred following M C A O , evidenced by forelimb flexion and muscle weakness, was greater in SHR (neurological grade = 2.0 +_ 0. I) than SD (1.0 + 0.4) or WKY (0.7 _+ 0.4) rats (N = 7-9, p < 0.05). SHR also exhibited sensory motor deficits following M C A O compared to sham-operation, with decreased normal placement response of the hindlimb (07o normal = 20 vs. 83, N = 23-30, p decreased rota-rod (41 ::t: 7 vs. 126 _+ 19 on rod, N = 10-15, p < 0.05) and balance beam (25 _+ 5 vs. 1 16 _+ 29 s on beam, N = 5-7, p < 0.05) performance. However, an index of general motor activity was not affected by permanent M C A O . Triphenyltetrazolium-stained forebrain tissue analyzed by planimetry revealed a significantly larger and more consistent cortical infarction in SHR (hemispheric infarction = 27.9 +_ 1.5°70) compared to SD (15.4 _+ 4.1070) and WKY (4.0 +_ 2.4070) rats (N = 7-9, p < 0.05), occupying predominantly the frontal and parietal areas. Also, a significant degree of ipsilateral hemispheric swelling (4.6 _+ 0.9070, N = 7-9, p < 0.05) and increased brain water content (78.4 _+ 0.3070 to 80.4 _+ 0.2070, N = 8-9, p < 0.05) was identified in SHR that was not observed in SD or WKY rats. A novel model of temporary M C A O also was evaluated in the hypertensive and normotensive rat strains. Initially, the effect of increasing MCAO-time followed by 24 h reperfusion in SHR was studied. During temporary M C A O (20 to 300 min), persistent and stable decreases in local microvascular perfusion (i.e., to 15-20070 of pre-MCAO levels) were verified in the primary ischemic zones of the cortex. U p o n reperfusion of the artery, local microvascular perfusion returned immediately to pre-MCAO levels and remained at those levels. An occlusion-time-related increase in the degree of cortical infarction, hemispheric swelling and neurological deficits was observed (N = 6-8 per timepoint, p < 0.05). Following temporary M C A O , the largest cortical infarcts and neurological deficits (i.e., the m a x i m u m effects under these conditions) were produced by 160 min M C A O with reperfusion. Using these o p t i m u m parameters, SD (6.7 _+ 2.0070) and WKY (1.4 _+ 0.8070) rats exhibited significantly smaller cortical infarctions than SHR (13.2 +_ 2.0070, N = 6-7, p < 0.05). The susceptibility to global and focal ischemia of SHR compared to normotensive rats was discussed in detail. Specifically, the increased SHR neuropathology following ischemia and the potential mechanisms for these effects in genetic hypertension was evaluated based on the available literature/data. Although, much information suggests that SHR are a useful model of focal ischemia (i.e., the histopathological and functional consequences due to M C A O are large and highly reproducible), SHR do exhibit significant differences in the morphology of cerebral blood vessels that impact on functional collateral cerebral circulation and its regulation. Factors such as increased arterial vessel wall thickness and reduced internal diameter, increased collateral vessel resistance and impaired vasodilation reserve can limit collateral blood flow and predispose SHR to cerebral ischemia and infarction. Also, an increased number a n d / o r sensitivity of perivascular mononuclear leukocytes and associated cytokine release is observed in the cerebral circulation of SHR. These cytokines (e.g., T u m o r Necrosis Factor-a) can predispose the vascular endothelium to a procoagulant surface m e m b r a n e so that ischemia can more readily provoke local vessel obstruction a n d / o r hemorrhage. These appear to be important mechanistic factors contributing to increased SHR ischemic damage compared to normotensive animals. This increased susceptibility/sensitivity of hypertensive rats to the effects of ischemia appears to underlie hypertension as a risk factor in focal stroke. Focal ischemia Spontaneously hypertensive rats Blood flow Perivascular mononuclear leukocytes
Normotensive rats
Stroke
Collateral circulation
L Presented, in part, at the First International Conference on Stroke, Geneva, Switzerland, May 30-June 1, 1991. 219
220
BARONE ET AL.
HYPERTENSION is the single most important risk factor for stroke in man (29,46,64), with cerebral infarction reported more frequently in the clinic for hypertensive individuals than for those with normal blood pressure (67). The animal model of chronic hypertension that shares many characteristics with human hypertension (32) is the spontaneously hypertensive rat (SHR), developed by systematic inbreeding of rats of the Wistar Kyoto (WKY) strain that had slightly elevated blood pressure (92). The stroke-prone SHR strain was derived from SHR. SHR do not exhibit spontaneous stroke. However, in stroke-prone SHR, both cerebral hemorrhages and infarcts occur spontaneously in greater than 80% of the males (93). Several experimental studies have suggested strongly that SHR and stroke-prone SHR exhibit more severe neuropathologic effects than normotensive animals following global or focal" cerebral ischemia. For example, following bilateral common carotid artery occlusion (i.e., global forebrain ischemia), diffuse and extensive cerebral infarcts occur very frequently in SHR, with normotensive rats exhibiting only occasional, small, well-circumscribed infarcts (90,95). Under these conditions, hypertensive animals suffer from an increased blood pressure drop distal to the carotid ligation (38,40,42), greater mortality (39,66), increase anaerobic metabolites with a larger decrease in adenosine triphosphate (36,39,58), and larger decreases in regional blood flow (13,37,38,58) than normotensive rats. SHR also exhibit significant cerebral ischemia during hemorrhagic hypotension compared to normotensive rats (119). An increased SHR susceptibility to cerebral ischemia also has been suggested in models of focal cerebral ischemia. In several different models of permanent middle cerebral artery occlusion (MCAO), SHR and stroke-prone SHR tend to require less extensive ischemic insult to produce infarctions and exhibit larger cerebral infarctions than normotensive rats (3,5,10,16,18,28,47,109). Rat models of MCAO are useful approximations of the ischemic hemispheric infarctions that occur in humans. However, models of rat focal ischemia are variable and depend on strain and surgical procedures [see (44) for review]. Since SHR generally appear to be more sensitive to cerebral ischemia, these animals provide an interesting strain to help understand risk factors in stroke. However, a systematic comparison of the effects of focal ischemia, especially focal ischemia with reperfusion, a recently established variant of the MCAO model (3), in hypertensive compared to normotensive rat strains using the same microsurgical procedures should be made. In order to make these comparisons, it is necessary to complete a detailed evaluation/characterization of the neurological deficits and cerebral ischemic damage making direct comparison of SHR versus normotensive strains following the exact same experimental focal ischemia with and without reperfusion. The purpose of the present review is to 1) discuss the sensitivity of SHR to cerebral ischemia, 2) to systematically study/compare the sensitivity of SHR to ischemia by a detailed neurological and histopathologic comparison of SHR to two normotensive strains, [i.e., Sprague-Dawley (SDR) and WKY rats] following permanent MCAO and MCAO with reperfusion, and 3) to interpret the data in reference to available related literature on SHR in order to enhance our understanding of the mechanism(s) for hypertension as a risk factor in focal stroke. METHOD Animals Three strains of mature male rats (SHR, SD and WKY) were obtained from commercial vendors (Taconic Farms, Ger-
mantown, NY; Charles River, Danvers, MA; and Charles River, respectively) at 18 wk of age (250-300 g in weight) and were housed for 2 to 4 weeks prior to utilization in these studies. In order to verify that the strains of animals studied were indeed hypertensive and normotensive, groups of animals from each strain were anesthetized with 2% isoflourane (Anaquest, Madison, WI) and chronically prepared under aseptic conditions for recording of blood pressure. The femoral artery was cannulated with polyethylene tubing (PE60; Clay Adams, Parsippany, N J) extending just into the descending aorta. The tubing was lead subdermally from the artery and exteriorized between the scapula just below the back of the neck and cleared/ filled with sterile isotonic saline. Incisions were closed using 2-0 silk suture and treated with 5% lidocaine ointment (Astra Pharmaceuticals, Westborough, MA). Animals recovered from surgery/anesthesia within 5 min. Mean arterial blood pressures were recorded 4 to 5 h after surgery for 5 min/rat by connecting the exteriorized tubing in each rat to a Statham pressure transducer (P23Db; Statham Medical Instruments, Los Angeles, CA) with output to a polygraph (Model R711; Beckman Instruments, Inc., Fullerton, CA). Focal Stroke Procedure MCAO or sham surgery was carried out in the SHR, SD and WKY rats under sodium pentobarbital (65 mg/kg, i.p. and supplemented as needed) anesthesia. All animals were allowed free access to food and water prior to and after surgery. Body temperature was maintained at 37°C using a heating pad throughout the surgical procedure. Surgery was conducted similar to that described previously (2,4). The right dorsal surface of the head was shaved and prepped with providone-iodine, and the rat placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA) with the surgery (right) side of the head superior. A 1-2 cm incision was made between the orbit and the external auditory canal. The temporal muscle was dissected from the skull and retracted without damaging the zygomatic bone or mandibular nerve. Under an operating microscope and with saline irrigation, a 2-3 mm craniotomy was made just rostral to the zygomatic-squamosal skull suture. The dura was opened over the artery using the modified tip of a 30-gauge needle. The hooked tip of a teflon coated platinum-irridium wire (0.0045 inch diameter; Medwire, Mount Vernon, NY) that was mounted on a micromanipulator was placed under the MCA and the artery was pulled 0.5-1.0 mm away from the brain surface for occlusion. For permanent right MCAO, using electrocoagulation (Force 2 Electrosurgical Generator, Valley Lab Inc., Boulder, CO), the artery was simultaneously occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein. For temporary right MCAO, the artery was lifted away from the brain surface at the same level, enough to occlude blood flow (i.e., as indicated from cortical microvascular perfusion monitored during the procedure; see below) for 0-300 min and then reperfused for 24 h. Some animals were reperfused for 5 days in order to determine if further damage developed beyond 24 h. Since differences have been identified following left compared to right MCAO (48,104,105) we utilized only right MCAO in the present studies. In sham-operated animals (i.e., 0 min MCAO always in SHR rats), the dura was opened over the right artery but the artery was not occluded. A small piece of sterile saline-soaked Gelfoam (Upjohn, Kalamazoo, MI) was then positioned over the craniotomy and the temporalis muscle and skin were closed in two layers. Animals were allowed to recover from anesthesia under a heating lamp and then were returned to their cages.
HYPERTENSION AND ISCHEMIC SENSITIVITY Laser-Doppler flowmetry was used to verify occlusion and reperfusion of the MCAO by monitoring local cortical microvascular perfusion in the primary ischemic zone of the cortex receiving blood supply from the MCA. To this end, a 2-3 mm diameter hole was drilled through the skull above the cortical area receiving blood supply from the MCA [i.e., centered at AP = 0 mm and L = 5 mm from bregma with level skull according to (97)]. The probe (1 mm diameter) of a LaserDoppler perfusion monitor (Periflux PF3; Perimed, Stockholm, Sweden) then was positioned on the dural surface and the local cortical perfusion was monitored before and after MCAO, and during MCA reperfusion. The calibrated output of the perfusion monitor was connected to a Beckman R711 polygraph.
Monitoring Neurological/Motor Function Prior to histologic processing of forebrain tissue 24 h post MCAO, a neurologic examination that included several tests was performed and the severity of deficits due to sham or MCAO surgery was determined. Each animal was classified using an established "Neurological Grade" procedure (5) into one of four grades: Grade 0 (no observable deficit), Grade 1 (any amount of consistent contralateral forelimb flexion), Grade 2 (reduced resistance to lateral push toward the paretic, contralateral side) or Grade 3 (circling behavior toward the paretic side). The Neurologic Grade quantitates the contralateral hemiparalysis and hemiparesis that can be a consequence of focal ischemia and the associated ipsilateral hemispheric infarct. A "Hindlimb Placement Test" was also performed by holding each rat with its face away from the edge of a table while the contralateral hindlimb is pulled over the edge of the table and extended downward. A normal response, observed in non-surgically treated or sham-operated animals or ipsilateral to the cerebral surgery, is an immediate placement of the hindlimb back onto the table. An abnormal response is no limb placement/movement. "Rota-Rod" testing was carried out prior to and after surgery. In this test, prior to surgery each rat is placed on a rota-rod (diameter 3 cm) treadmill for rats (Hugo Sachs Elektronik KG, March-Hugstetten, Germany) at 10 rpm and a criterion of 60 s remaining on the treadmill is established. After surgery, the duration (in s) each rat remained on the rota-rod is measured. "Balance Beam" testing also was carried out before and after surgery. Similarly in this test, prior to surgery each rat is placed on a horizontally positioned wooden rod (60 cm long and 2 cm in diameter), and a criterion of 60 s on the beam is established. After surgery, the duration (in seconds) each rat remained on the beam is measured. Finally "motor activity" testing was carried out before and after surgery. In this test, each rat is placed in a 2 ft 2 enclosure with two intersecting lines that define four separate 1 ft 2 areas. The number of times both forepaws cross a line is recorded for a 3 min period.
Measurements of&chemic Damage Following the neurologic evaluation (24 h after surgery) rats were euthanized with an overdose of sodium pentobarbital. Within 2-3 min, brains were removed and six coronal forebrain slices (2 mm thick) were made from the level of the olfactory bulbs to the cortical-cerebellar junction using a rat brain slicer [(59); Zivic-MiUer Laboratories Inc., Allison Park, PA]. These forebrain slices then were immersed immediately in a 1% solution of triphenyltetrazolium chloride (TTC) in phosphate buffer at 37°C for 20-30 min (6,78). Stained tissues then were fixed by infiltration in 10% phosphate buf-
221 fered formalin. The two sides of each TTC-stained section were photographed in color using a polaroid camera. These photographs were analyzed for the quantification of ischemic damage using an image analysis system (Amersham RAS 3000; Loats Associates, Inc.). Morphological changes following surgery were evaluated in the entire forebrain (total of 11 planar surfaces) for each animal. The 11 planar images were obtained from each side of the six 2 mm thick sections and correspond approximately to 1 mm section surfaces from + 5mm to - 5mm from bregma (97) and include the complete forebrain. These planar image surfaces (from the photographs) were digitized and used in the Image Analysis System for planimetry determination of infarct size and swelling. Two parameters of ischemic damage due to MCAO were determined for each slice as described previously (2,4,98,122). "Hemispheric swelling" was expressed as the percent increase in size of the ipsilateral (i.e., surgery side) hemisphere over the contralateral (normal) hemisphere and was calculated as:
Percent Hemispheric = Swelling
Ipsilateral Contralateral Hemisphere - Hemisphere Area Area Contralateral Hemisphere Area
X
100
"Infarct size" which was expressed as the percent infarcted tissue in reference to the contralateral (normal) hemisphere and was calculated as: Percent Hemispheric = Infarct Size
Infarct area x 100 Contralateral Hemispheric Area
The swelling and infarct size were expressed in reference to the contralateral hemisphere (i.e., ipsilateral ischemic damage was normalized to the normal contralateral hemisphere). These parameters were determined for each slice to evaluate the profile of damage throughout the forebrain (i.e., "forebrain profile") and for "total" forebrain changes by using the sum of all individual slice data in these formulas. The occurrence of brain edema associated with hemispheric swelling following MCAO was determined by comparison of wet/dry weight as described previously (45,118). Rats were sacrificed by an overdose of sodium pentobarbital 24 h after sham or MCAO surgery. The brains were quickly removed, the forebrain isolated at the cerebellar cortical junction and cut into two hemispheres, and each forebrain hemisphere measured on a Mettler Type H5 chemical balance (Mettler Instruments Corp., Hightstown, N J) within 2 min after decapitation. The dry weight was measured on the same scale after drying the hemisphere in an oven at 80°C for 48-72 h. The water content of each hemisphere was calculated as the difference between the wet and dry weight as a percent fraction from the wet weight: Percent Wet Weight - Dry Weight = x 100 Water Content Wet Weight
Stat&tical Analysis All data are presented as mean + SEM. Comparisons between two groups were made for unpaired data using Student's t-test (53). In the case of nonparametric data (i.e., the Hind-
B A R O N E ET AL.
222
EFFECTS OF PERMANENT MCA OCCLUSION ON CORTICAL MICROVASCULAR PERFUSION
limb Pull Test p r o p o r t i o n s ) , the X z test for two i n d e p e n d e n t samples was utilized (107). C o m p a r i s o n s between m o r e t h a n two groups were m a d e using a one-way analysis o f variance with "Tukey a" a n d / o r " D u n n e t t " tests as a p p r o p r i a t e followup analyses (123). Differences were considered significant if p < 0.05. RESULTS
Pick-up MCA
Percent Basal
Mean Arterial Pressure The results o f conscious m e a n arterial b l o o d pressure recording in the three strains of rats are listed in Table 1. S H R rats exhibited a significantly increased b l o o d pressure compared to n o r m o t e n s i v e SD or WKY rats. These differences were large and as expected even 5 h after surgical p r e p a r a t i o n of the animals.
Permanent (24 h) MCA 0 Cortical microvascular perfusion. A decrease in local microvascular perfusion was verified in the p r i m a r y ischemic area o f the cortex following p e r m a n e n t M C A O . Local cortical perfusion m o n i t o r e d at the cortical surface was decreased perm a n e n t l y (i.e., between 15-20070 o f original baseline a n d verified for recording periods greater t h a n 40 rain) following M C A O . In rats with completely artifact free Laser-Doppler flowmetry recordings (N = 13), a m a i n t a i n e d decrease to 15.6 _+ 1.7°70 o f the pre-occlusion basal perfusion level was measured (Fig. 1). No differences in perfusion of the p r i m a r y / central ischemic cortex were observed before a n d after M C A O between the three strains. Neurological deficits. Deficits were d e m o n s t r a t e d in the Neurological E x a m i n a t i o n test series 24 h following permanent M C A O . Tests usually were c o n d u c t e d with the experimenter blind to t r e a t m e n t a n d the reliability o f test results between different experimenters had been d e m o n s t r a t e d previously (data not shown). The "Neurological G r a d e " indicated that M C A O in all strains p r o d u c e d some degree o f contralateral forepaw hemiparalysis a n d hemiparesis indicated typically by a grade o f 2 in S H R rats c o m p a r e d to a grade typically o f zero in s h a m - o p e r a t e d animals (Table 2). It was unusual to see any animal circle in the contralateral direction following M C A O surgery or to exhibit any observable deficit following sham surgery. However, in a h e a d - t o - h e a d c o m p a r i s o n S H R did exhibit a significantly greater neurological grade t h a n SD or WKY rats (Fig. 2), indicating an increased neurological deficit ( p deficits in several additional tests o f s e n s o r y - m o t o r function after S h a m or p e r m a n e n t M C A O surgery). The results of these tests also are listed in Table 2. In the " H i n d l i m b P l a c e m e n t " test, s h a m - o p e r a t e d animals typically exhibit a n o r m a l limb placement. Following M C A O , significantly more
Perfus~on
~ Occlude MCA
100 t 50 0 I
Srain
i
FIG. 1. Example of decreased microvascular perfusion recorded/verified in core ischemic cortext area before and after permanent MCAO. Cortical perfusion after MCAO typically decreased greater than 80% of pre-occlusion baseline. The maintained/permanent decrease for 13 animals with artifact free recordings was to 15.6 + 1.7°70of baseline. Similar effects were seen in all rat strains studied.
TABLE 2 N E U R O L O G I C A L DEFICITS IN SHR
Test +
Sham (N)
Hindlimb Placement (o70 normal) 82.6 (23) Roto-Rod (sec) 126 + 19(10) Balance Beam (sec) 116 _+ 29 (5) Motor Activity** (crossings) 16 + I t o 1 5 + 1(13)
Permanent MCAO (N)
20.0 (30)* 41 _ 7 (15)* 25 _+ 5 (7)* 17 _ I to 14 + I (24)
+Testing procedures and quantitation as described in text. *Significantly different from Sham, p < 0.05. **Line crossings before to after Sham or MCAO surgery.
Permanent MCAO Neurological Grade 2.5
2.0
Grade Following Surgery
1.5
1.0
TABLE 1 M E A N A R T E R I A L PRESSURE IN C O N S C I O U S RATS
Strain
N
Mean Arterial Pressure*
SHR SD WKY
5 4 5
161.6 +_ 3.4** 123.3 _+ 6.2 121.0 _+ 4.3
0.5
0.0
*Preparation of animals and testing as described in text. **Significantly increased above SD and WKY rats; p < 0.05.
Sham
Spontaneously Hypertensive
Sprague Oawley
Wlstar Kyoto
FIG. 2. Neurological Grades in Sham (SHR: N = 8)-operated and permanent MCAO-operated rats from three strains 24 h post-surgery. Significantly larger neurological deficits were observed for SHR (N = 9) compared to normotensive SD (N = 8) and WKY (N = 7) rats. * = significantly, p < 0.05, different from Sham; + = significantly, p < 0.05, different from SD rats.
HYPERTENSION
AND ISCHEMIC SENSITIVITY
S H R failed to exhibit a n o r m a l limb placement response ( p < 0.05). Pre-test results for b o t h the "Neurological G r a d e " a n d the " H i n d l i m b P l a c e m e n t " test were always (0 _+ 0 a n d 100%0 in any group(s) o f animals tested (data not shown). F u r t h e r m o r e , p e r f o r m a n c e in the " R o t a - R o d " a n d the stationary "Balance B e a m " tests were significantly ( p < 0.05) decreased following M C A O c o m p a r e d to s h a m surgery, while the index o f m o t o r activity was not affected by M C A O compared to s h a m surgery. Brain infarct analysis. Figure 3 illustrates a typical infarct p r o d u c e d by p e r m a n e n t M C A O in S H R , as c o m p a r e d to S h a m surgery, identified 24 h after surgery following staining with T T C . These infarctions c o r r e s p o n d to volumes ranging from 128 to 179 m m 3 a n d typically were restricted to the cortex in the frontal and parietal areas. I n f a r c t i o n was not observed in S h a m (SHR) animals. Figure 4 illustrates the forebrain profile of infarction for s h a m (SHR) a n d the three strains o f M C A O - o p e r a t e d animals. No cortical infarctions were observed following sham surgery. In S H R , the infarction occupied a large p o r t i o n o f the a n t e r i o r forebrain (i.e., 20 to 4 0 % o f the n o r m a l contralateral hemisphere), but decreased in size in the more posterior f o r e b r a i n areas. A m u c h smaller profile (i.e., less t h a n 2 0 % o f the n o r m a l contralateral hemisphere) was observed in the n o r m o t e n s i v e strains, with WKY rats exhibiting the smallest infarct profile. Figure 5 illustrates the total hemispheric infarct calculated from the sum of the slice data. S H R exhibited the largest and least variable total hemispheric infarcts following p e r m a n e n t M C A O (p < 0.05). Brain swelling analysis. The swelling measure for S h a m , S H R , a n d n o r m o t e n s i v e rats was variable. For s h a m - o p e r a t e d and n o r m o t e n s i v e animals it varied a r o u n d zero t h r o u g h o u t the forebrain. Following M C A O in S H R , the size of the ipsilateral hemisphere was increased, not necessarily related to
223
Permanent MCAO Forebrain Infarction Profile 50 ¸ A. Sham Surgery ~
I'~
"E m
.
o
v
$
30
~:
2o
B. Sponlaneously Hypertensive C. Sprague Dawley
4O ~
D. Wlstar KyoIo
E
4
3
2 1 0 -1 -2 -3 -4 Distance From Bregma (ram)
-5
FIG. 4. Profile of Forebrain Hemispheric Infarction for sham (SHR; N = 8)- and permanent MCAO-operated rats from three strains (SHR; N = 9; SD; N = 8; WKY; N = 7) 24 h post-surgery. Measures were determined using image analysis/planimetry and expressed in reference to the normal contralateral hemisphere as described in methods and Fig. 3.
SHAM SURGERY
MCAO SURGERY
! a,
FIG. 3. Photographs of forebrain sections stained with triphenyltitrazolium 24 h after Sham surgery (left) or MCAO surgery (right). Eleven planar images were obtained from each side of six 2 mm thick sections of the complete forebrain and correspond approximately to I mm section surfaces from -b 5 mm to - 5 mm (i.e., from left to right, top to bottom) in reference to bregma 09). These planar images from the photographs were used in the Image Analysis System for planimetry determinations of infarct size and swelling.
224
B A R O N E E T AL. Permanent
Permanent MCAO Total Hemisheric Water Content
MCAO
Total Hemispheric I n f a r c t i o n 81.0
x
30
+ Contralateral 80.5
25
Total Percent Hemispheric
Ipsda '~'~
80.0,
20
Infarction
E EL
10
78.0,
+
77.5, Sham
Sham
Spontaneously Spragul Hypertensive Dawley
Wislar
Kyoto
FIG. 5. Total Hemispheric Infarction for sham (SHR; N = 9) and permanent MCAO-operated rats from three strains 24 h post-surgery. Measures determined as described in methods and Fig. 3. Significantly larger forebrain infarctions were observed for SHR (N = 9) compared to SD (N = 8) and WKY (N = 7) rats. * =significantly, p < 0.05, different from Sham; + = significantly, p < 0.05, different from SD rats.
the degree of infarction but primarily in the more a n t e r i o r a n d more posterior aspects of the forebrain. The total hemispheric swelling calculated from the sum of the slice data is illustrated in Fig. 6 S H R exhibited a significant increase in hemispheric size that was not o b t a i n e d in S h a m - o p e r a t e d S H R or n o r m o tensive animals ( p < 0.05). Figure 7 illustrates the significant
Total
Permanent MCAO Hemispheric Swelling
6-
=k
5
1
4 3 2-
-1-2-
-3 ~
79.0. 78.5,
5
0
79.5.
o
15
Percent Change Ipsilateral Hemispheric Size
BB
Sham
Spontaneously Hyparlenslve
Sptague Dawley
Wlstar Kyoto
FIG. 6. Total Hemispheric Swelling for sham (SHR)- and permanent MCAO-operated rats from three strains 24 h post-surgery. Percent change in ipsilateral hemispheric size was determined as described in the methods and Fig. 3. A significant increase in ipsilateral hemispheric size was observed in SHR MCAO-surgery animals (N = 9) that was not observed in sham (N = 8), SD (N = 8) or WKY (N = 7) rats. * = significantly, p < 0.05, different from Sham.
MCAO
Surgery FIG. 7. Percent water content for ipsilateral and contralateral hemispheres in sham (N = 8)- and MCAO (N = 9)-operated SHR 24 h post-surgery. A significant increase (*; p < 0.01) in the ipsilateral hemispheric water content (i.e., edema) occurred following MCAO but not following sham surgery.
increase in ipsilateral hemispheric water content that occurs 24 h after M C A O (p < 0.01) in SHR. No change in hemispheric water content was produced by sham surgery.
Temporary MCA 0 with Reperfusion (24 h) in SHR Cortical microvascular perfusion. Initial studies were carried out to characterize the effects of M C A O with reperfusion in SHR in order to select the a p p r o p r i a t e occlusion-time parameter for c o m p a r i s o n to n o r m o t e n s i v e animals. As for perm a n e n t M C A O , a similar decrease in local microvascular perfusion was verified in the primary ischemic cortical area o f S H R during t e m p o r a r y M C A O . Figure 8 illustrates these results for several occlusion times that were studied. During M C A O , cortex perfusion was decreased persistently for the total d u r a t i o n of the controlled occlusion. Also, u p o n reperfusion regional cortical blood flow recovered to pre-occlusion levels. In some animals cortical blood flow was recorded for periods of up to 60 rain post-reperfusion a n d cortical perfusion was observed to remain at p r e - M C A O levels. A similar pattern of cortical blood flow changes in the primary ischemic area (i.e., a decrease followed by a complete recovery o f perfusion) was observed in n o r m o t e n s i v e animals during M C A O with reperfusion (data not shown). Neurological deficits. Figure 9 illustrates Neurological G r a d e results for t e m p o r a r y M C A O of 0 min (sham surgery) to 300 min followed by 24 h reperfusion a n d for p e r m a n e n t (24 h) M C A O in SHR. The Neurological G r a d e was very sensitive to even short periods of t e m p o r a r y M C A O . A n occlusion-time-related increase in neurological deficit was observed (p < 0.05). Occlusion-times of 40 rain and a b o v e significantly increased the grade, with m a x i m u m grades (i.e. similar to that which occurs following p e r m a n e n t M C A O ) achieved after 160 min o f t e m p o r a r y M C A O . Neuropathological changes. Figure 10 illustrates the forebrain profile o f infarction for several periods o f t e m p o r a r y occlusion. A clear ischemia-time-related increase in the forebrain infarct profile was observed, with the largest infarct profile observed following p e r m a n e n t M C A O . Figure 11 illus-
HYPERTENSION
AND
ISCHEMIC
SENSITIVITY
225
Ischemic Cortex Microvascular Perfusion
MCAO Time in SHR Forebrain Infarction Profile 40
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0149-7634/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd.
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Genetic Hypertension and Increased Susceptibility to Cerebral Ischemia 1 F.
C.
BARONE,
W.
J.
PRICE,
R.
F.
WHITE,
R.
N.
WlLLETTE
AND
G.
Z.
FEUERSTEIN
D e p a r t m e n t o f P h a r m a c o l o g y , S m i t h K l i n e B e e c h a m Pharmaceuticals, plc, K i n g o f Prussia, P A 19406 Received 5 August
1991
B A R O N E , F. C., W. J. PRICE, R. F. W H I T E , R. N. W I L L E T T E A N D G. Z. FEUERSTEIN. Genetic hypertension and increased susceptibility to cerebral ischemia. NEUROSCI BIOBEHAV REV 16(2) 219-233, 1 9 9 2 . - A review of the sensitivity of genetically hypertensive rats to cerebral ischemia was presented together with original data describing the systematic comparison of the effects of focal ischemia (permanent and temporary with reperfusion) performed in hypertensive and normotensive rats (i.e., blood pressures verified in conscious instrumented rats). Microsurgical techniques were used to isolate and occlude the middle cerebral artery (MCAO) of spontaneously hypertensive (SHR), Sprague-Dawley (SD) and Wistar Kyoto (WKY) rats at the level of the inferior cerebral vein. Following permanent (24 h) M C A O , persistent and similar decreases in local microvascular perfusion (i.e., to 15.6 _+ 1.7070 of pre-MCAO levels) were verified in the primary ischemic zone of the cortex for all strains using Laser-Doppler flowmetry. A contralateral hemiplegia that occurred following M C A O , evidenced by forelimb flexion and muscle weakness, was greater in SHR (neurological grade = 2.0 +_ 0. I) than SD (1.0 + 0.4) or WKY (0.7 _+ 0.4) rats (N = 7-9, p < 0.05). SHR also exhibited sensory motor deficits following M C A O compared to sham-operation, with decreased normal placement response of the hindlimb (07o normal = 20 vs. 83, N = 23-30, p decreased rota-rod (41 ::t: 7 vs. 126 _+ 19 on rod, N = 10-15, p < 0.05) and balance beam (25 _+ 5 vs. 1 16 _+ 29 s on beam, N = 5-7, p < 0.05) performance. However, an index of general motor activity was not affected by permanent M C A O . Triphenyltetrazolium-stained forebrain tissue analyzed by planimetry revealed a significantly larger and more consistent cortical infarction in SHR (hemispheric infarction = 27.9 +_ 1.5°70) compared to SD (15.4 _+ 4.1070) and WKY (4.0 +_ 2.4070) rats (N = 7-9, p < 0.05), occupying predominantly the frontal and parietal areas. Also, a significant degree of ipsilateral hemispheric swelling (4.6 _+ 0.9070, N = 7-9, p < 0.05) and increased brain water content (78.4 _+ 0.3070 to 80.4 _+ 0.2070, N = 8-9, p < 0.05) was identified in SHR that was not observed in SD or WKY rats. A novel model of temporary M C A O also was evaluated in the hypertensive and normotensive rat strains. Initially, the effect of increasing MCAO-time followed by 24 h reperfusion in SHR was studied. During temporary M C A O (20 to 300 min), persistent and stable decreases in local microvascular perfusion (i.e., to 15-20070 of pre-MCAO levels) were verified in the primary ischemic zones of the cortex. U p o n reperfusion of the artery, local microvascular perfusion returned immediately to pre-MCAO levels and remained at those levels. An occlusion-time-related increase in the degree of cortical infarction, hemispheric swelling and neurological deficits was observed (N = 6-8 per timepoint, p < 0.05). Following temporary M C A O , the largest cortical infarcts and neurological deficits (i.e., the m a x i m u m effects under these conditions) were produced by 160 min M C A O with reperfusion. Using these o p t i m u m parameters, SD (6.7 _+ 2.0070) and WKY (1.4 _+ 0.8070) rats exhibited significantly smaller cortical infarctions than SHR (13.2 +_ 2.0070, N = 6-7, p < 0.05). The susceptibility to global and focal ischemia of SHR compared to normotensive rats was discussed in detail. Specifically, the increased SHR neuropathology following ischemia and the potential mechanisms for these effects in genetic hypertension was evaluated based on the available literature/data. Although, much information suggests that SHR are a useful model of focal ischemia (i.e., the histopathological and functional consequences due to M C A O are large and highly reproducible), SHR do exhibit significant differences in the morphology of cerebral blood vessels that impact on functional collateral cerebral circulation and its regulation. Factors such as increased arterial vessel wall thickness and reduced internal diameter, increased collateral vessel resistance and impaired vasodilation reserve can limit collateral blood flow and predispose SHR to cerebral ischemia and infarction. Also, an increased number a n d / o r sensitivity of perivascular mononuclear leukocytes and associated cytokine release is observed in the cerebral circulation of SHR. These cytokines (e.g., T u m o r Necrosis Factor-a) can predispose the vascular endothelium to a procoagulant surface m e m b r a n e so that ischemia can more readily provoke local vessel obstruction a n d / o r hemorrhage. These appear to be important mechanistic factors contributing to increased SHR ischemic damage compared to normotensive animals. This increased susceptibility/sensitivity of hypertensive rats to the effects of ischemia appears to underlie hypertension as a risk factor in focal stroke. Focal ischemia Spontaneously hypertensive rats Blood flow Perivascular mononuclear leukocytes
Normotensive rats
Stroke
Collateral circulation
L Presented, in part, at the First International Conference on Stroke, Geneva, Switzerland, May 30-June 1, 1991. 219
220
BARONE ET AL.
HYPERTENSION is the single most important risk factor for stroke in man (29,46,64), with cerebral infarction reported more frequently in the clinic for hypertensive individuals than for those with normal blood pressure (67). The animal model of chronic hypertension that shares many characteristics with human hypertension (32) is the spontaneously hypertensive rat (SHR), developed by systematic inbreeding of rats of the Wistar Kyoto (WKY) strain that had slightly elevated blood pressure (92). The stroke-prone SHR strain was derived from SHR. SHR do not exhibit spontaneous stroke. However, in stroke-prone SHR, both cerebral hemorrhages and infarcts occur spontaneously in greater than 80% of the males (93). Several experimental studies have suggested strongly that SHR and stroke-prone SHR exhibit more severe neuropathologic effects than normotensive animals following global or focal" cerebral ischemia. For example, following bilateral common carotid artery occlusion (i.e., global forebrain ischemia), diffuse and extensive cerebral infarcts occur very frequently in SHR, with normotensive rats exhibiting only occasional, small, well-circumscribed infarcts (90,95). Under these conditions, hypertensive animals suffer from an increased blood pressure drop distal to the carotid ligation (38,40,42), greater mortality (39,66), increase anaerobic metabolites with a larger decrease in adenosine triphosphate (36,39,58), and larger decreases in regional blood flow (13,37,38,58) than normotensive rats. SHR also exhibit significant cerebral ischemia during hemorrhagic hypotension compared to normotensive rats (119). An increased SHR susceptibility to cerebral ischemia also has been suggested in models of focal cerebral ischemia. In several different models of permanent middle cerebral artery occlusion (MCAO), SHR and stroke-prone SHR tend to require less extensive ischemic insult to produce infarctions and exhibit larger cerebral infarctions than normotensive rats (3,5,10,16,18,28,47,109). Rat models of MCAO are useful approximations of the ischemic hemispheric infarctions that occur in humans. However, models of rat focal ischemia are variable and depend on strain and surgical procedures [see (44) for review]. Since SHR generally appear to be more sensitive to cerebral ischemia, these animals provide an interesting strain to help understand risk factors in stroke. However, a systematic comparison of the effects of focal ischemia, especially focal ischemia with reperfusion, a recently established variant of the MCAO model (3), in hypertensive compared to normotensive rat strains using the same microsurgical procedures should be made. In order to make these comparisons, it is necessary to complete a detailed evaluation/characterization of the neurological deficits and cerebral ischemic damage making direct comparison of SHR versus normotensive strains following the exact same experimental focal ischemia with and without reperfusion. The purpose of the present review is to 1) discuss the sensitivity of SHR to cerebral ischemia, 2) to systematically study/compare the sensitivity of SHR to ischemia by a detailed neurological and histopathologic comparison of SHR to two normotensive strains, [i.e., Sprague-Dawley (SDR) and WKY rats] following permanent MCAO and MCAO with reperfusion, and 3) to interpret the data in reference to available related literature on SHR in order to enhance our understanding of the mechanism(s) for hypertension as a risk factor in focal stroke. METHOD Animals Three strains of mature male rats (SHR, SD and WKY) were obtained from commercial vendors (Taconic Farms, Ger-
mantown, NY; Charles River, Danvers, MA; and Charles River, respectively) at 18 wk of age (250-300 g in weight) and were housed for 2 to 4 weeks prior to utilization in these studies. In order to verify that the strains of animals studied were indeed hypertensive and normotensive, groups of animals from each strain were anesthetized with 2% isoflourane (Anaquest, Madison, WI) and chronically prepared under aseptic conditions for recording of blood pressure. The femoral artery was cannulated with polyethylene tubing (PE60; Clay Adams, Parsippany, N J) extending just into the descending aorta. The tubing was lead subdermally from the artery and exteriorized between the scapula just below the back of the neck and cleared/ filled with sterile isotonic saline. Incisions were closed using 2-0 silk suture and treated with 5% lidocaine ointment (Astra Pharmaceuticals, Westborough, MA). Animals recovered from surgery/anesthesia within 5 min. Mean arterial blood pressures were recorded 4 to 5 h after surgery for 5 min/rat by connecting the exteriorized tubing in each rat to a Statham pressure transducer (P23Db; Statham Medical Instruments, Los Angeles, CA) with output to a polygraph (Model R711; Beckman Instruments, Inc., Fullerton, CA). Focal Stroke Procedure MCAO or sham surgery was carried out in the SHR, SD and WKY rats under sodium pentobarbital (65 mg/kg, i.p. and supplemented as needed) anesthesia. All animals were allowed free access to food and water prior to and after surgery. Body temperature was maintained at 37°C using a heating pad throughout the surgical procedure. Surgery was conducted similar to that described previously (2,4). The right dorsal surface of the head was shaved and prepped with providone-iodine, and the rat placed in a stereotaxic device (David Kopf Instruments, Tujunga, CA) with the surgery (right) side of the head superior. A 1-2 cm incision was made between the orbit and the external auditory canal. The temporal muscle was dissected from the skull and retracted without damaging the zygomatic bone or mandibular nerve. Under an operating microscope and with saline irrigation, a 2-3 mm craniotomy was made just rostral to the zygomatic-squamosal skull suture. The dura was opened over the artery using the modified tip of a 30-gauge needle. The hooked tip of a teflon coated platinum-irridium wire (0.0045 inch diameter; Medwire, Mount Vernon, NY) that was mounted on a micromanipulator was placed under the MCA and the artery was pulled 0.5-1.0 mm away from the brain surface for occlusion. For permanent right MCAO, using electrocoagulation (Force 2 Electrosurgical Generator, Valley Lab Inc., Boulder, CO), the artery was simultaneously occluded and cut dorsal to the lateral olfactory tract at the level of the inferior cerebral vein. For temporary right MCAO, the artery was lifted away from the brain surface at the same level, enough to occlude blood flow (i.e., as indicated from cortical microvascular perfusion monitored during the procedure; see below) for 0-300 min and then reperfused for 24 h. Some animals were reperfused for 5 days in order to determine if further damage developed beyond 24 h. Since differences have been identified following left compared to right MCAO (48,104,105) we utilized only right MCAO in the present studies. In sham-operated animals (i.e., 0 min MCAO always in SHR rats), the dura was opened over the right artery but the artery was not occluded. A small piece of sterile saline-soaked Gelfoam (Upjohn, Kalamazoo, MI) was then positioned over the craniotomy and the temporalis muscle and skin were closed in two layers. Animals were allowed to recover from anesthesia under a heating lamp and then were returned to their cages.
HYPERTENSION AND ISCHEMIC SENSITIVITY Laser-Doppler flowmetry was used to verify occlusion and reperfusion of the MCAO by monitoring local cortical microvascular perfusion in the primary ischemic zone of the cortex receiving blood supply from the MCA. To this end, a 2-3 mm diameter hole was drilled through the skull above the cortical area receiving blood supply from the MCA [i.e., centered at AP = 0 mm and L = 5 mm from bregma with level skull according to (97)]. The probe (1 mm diameter) of a LaserDoppler perfusion monitor (Periflux PF3; Perimed, Stockholm, Sweden) then was positioned on the dural surface and the local cortical perfusion was monitored before and after MCAO, and during MCA reperfusion. The calibrated output of the perfusion monitor was connected to a Beckman R711 polygraph.
Monitoring Neurological/Motor Function Prior to histologic processing of forebrain tissue 24 h post MCAO, a neurologic examination that included several tests was performed and the severity of deficits due to sham or MCAO surgery was determined. Each animal was classified using an established "Neurological Grade" procedure (5) into one of four grades: Grade 0 (no observable deficit), Grade 1 (any amount of consistent contralateral forelimb flexion), Grade 2 (reduced resistance to lateral push toward the paretic, contralateral side) or Grade 3 (circling behavior toward the paretic side). The Neurologic Grade quantitates the contralateral hemiparalysis and hemiparesis that can be a consequence of focal ischemia and the associated ipsilateral hemispheric infarct. A "Hindlimb Placement Test" was also performed by holding each rat with its face away from the edge of a table while the contralateral hindlimb is pulled over the edge of the table and extended downward. A normal response, observed in non-surgically treated or sham-operated animals or ipsilateral to the cerebral surgery, is an immediate placement of the hindlimb back onto the table. An abnormal response is no limb placement/movement. "Rota-Rod" testing was carried out prior to and after surgery. In this test, prior to surgery each rat is placed on a rota-rod (diameter 3 cm) treadmill for rats (Hugo Sachs Elektronik KG, March-Hugstetten, Germany) at 10 rpm and a criterion of 60 s remaining on the treadmill is established. After surgery, the duration (in s) each rat remained on the rota-rod is measured. "Balance Beam" testing also was carried out before and after surgery. Similarly in this test, prior to surgery each rat is placed on a horizontally positioned wooden rod (60 cm long and 2 cm in diameter), and a criterion of 60 s on the beam is established. After surgery, the duration (in seconds) each rat remained on the beam is measured. Finally "motor activity" testing was carried out before and after surgery. In this test, each rat is placed in a 2 ft 2 enclosure with two intersecting lines that define four separate 1 ft 2 areas. The number of times both forepaws cross a line is recorded for a 3 min period.
Measurements of&chemic Damage Following the neurologic evaluation (24 h after surgery) rats were euthanized with an overdose of sodium pentobarbital. Within 2-3 min, brains were removed and six coronal forebrain slices (2 mm thick) were made from the level of the olfactory bulbs to the cortical-cerebellar junction using a rat brain slicer [(59); Zivic-MiUer Laboratories Inc., Allison Park, PA]. These forebrain slices then were immersed immediately in a 1% solution of triphenyltetrazolium chloride (TTC) in phosphate buffer at 37°C for 20-30 min (6,78). Stained tissues then were fixed by infiltration in 10% phosphate buf-
221 fered formalin. The two sides of each TTC-stained section were photographed in color using a polaroid camera. These photographs were analyzed for the quantification of ischemic damage using an image analysis system (Amersham RAS 3000; Loats Associates, Inc.). Morphological changes following surgery were evaluated in the entire forebrain (total of 11 planar surfaces) for each animal. The 11 planar images were obtained from each side of the six 2 mm thick sections and correspond approximately to 1 mm section surfaces from + 5mm to - 5mm from bregma (97) and include the complete forebrain. These planar image surfaces (from the photographs) were digitized and used in the Image Analysis System for planimetry determination of infarct size and swelling. Two parameters of ischemic damage due to MCAO were determined for each slice as described previously (2,4,98,122). "Hemispheric swelling" was expressed as the percent increase in size of the ipsilateral (i.e., surgery side) hemisphere over the contralateral (normal) hemisphere and was calculated as:
Percent Hemispheric = Swelling
Ipsilateral Contralateral Hemisphere - Hemisphere Area Area Contralateral Hemisphere Area
X
100
"Infarct size" which was expressed as the percent infarcted tissue in reference to the contralateral (normal) hemisphere and was calculated as: Percent Hemispheric = Infarct Size
Infarct area x 100 Contralateral Hemispheric Area
The swelling and infarct size were expressed in reference to the contralateral hemisphere (i.e., ipsilateral ischemic damage was normalized to the normal contralateral hemisphere). These parameters were determined for each slice to evaluate the profile of damage throughout the forebrain (i.e., "forebrain profile") and for "total" forebrain changes by using the sum of all individual slice data in these formulas. The occurrence of brain edema associated with hemispheric swelling following MCAO was determined by comparison of wet/dry weight as described previously (45,118). Rats were sacrificed by an overdose of sodium pentobarbital 24 h after sham or MCAO surgery. The brains were quickly removed, the forebrain isolated at the cerebellar cortical junction and cut into two hemispheres, and each forebrain hemisphere measured on a Mettler Type H5 chemical balance (Mettler Instruments Corp., Hightstown, N J) within 2 min after decapitation. The dry weight was measured on the same scale after drying the hemisphere in an oven at 80°C for 48-72 h. The water content of each hemisphere was calculated as the difference between the wet and dry weight as a percent fraction from the wet weight: Percent Wet Weight - Dry Weight = x 100 Water Content Wet Weight
Stat&tical Analysis All data are presented as mean + SEM. Comparisons between two groups were made for unpaired data using Student's t-test (53). In the case of nonparametric data (i.e., the Hind-
B A R O N E ET AL.
222
EFFECTS OF PERMANENT MCA OCCLUSION ON CORTICAL MICROVASCULAR PERFUSION
limb Pull Test p r o p o r t i o n s ) , the X z test for two i n d e p e n d e n t samples was utilized (107). C o m p a r i s o n s between m o r e t h a n two groups were m a d e using a one-way analysis o f variance with "Tukey a" a n d / o r " D u n n e t t " tests as a p p r o p r i a t e followup analyses (123). Differences were considered significant if p < 0.05. RESULTS
Pick-up MCA
Percent Basal
Mean Arterial Pressure The results o f conscious m e a n arterial b l o o d pressure recording in the three strains of rats are listed in Table 1. S H R rats exhibited a significantly increased b l o o d pressure compared to n o r m o t e n s i v e SD or WKY rats. These differences were large and as expected even 5 h after surgical p r e p a r a t i o n of the animals.
Permanent (24 h) MCA 0 Cortical microvascular perfusion. A decrease in local microvascular perfusion was verified in the p r i m a r y ischemic area o f the cortex following p e r m a n e n t M C A O . Local cortical perfusion m o n i t o r e d at the cortical surface was decreased perm a n e n t l y (i.e., between 15-20070 o f original baseline a n d verified for recording periods greater t h a n 40 rain) following M C A O . In rats with completely artifact free Laser-Doppler flowmetry recordings (N = 13), a m a i n t a i n e d decrease to 15.6 _+ 1.7°70 o f the pre-occlusion basal perfusion level was measured (Fig. 1). No differences in perfusion of the p r i m a r y / central ischemic cortex were observed before a n d after M C A O between the three strains. Neurological deficits. Deficits were d e m o n s t r a t e d in the Neurological E x a m i n a t i o n test series 24 h following permanent M C A O . Tests usually were c o n d u c t e d with the experimenter blind to t r e a t m e n t a n d the reliability o f test results between different experimenters had been d e m o n s t r a t e d previously (data not shown). The "Neurological G r a d e " indicated that M C A O in all strains p r o d u c e d some degree o f contralateral forepaw hemiparalysis a n d hemiparesis indicated typically by a grade o f 2 in S H R rats c o m p a r e d to a grade typically o f zero in s h a m - o p e r a t e d animals (Table 2). It was unusual to see any animal circle in the contralateral direction following M C A O surgery or to exhibit any observable deficit following sham surgery. However, in a h e a d - t o - h e a d c o m p a r i s o n S H R did exhibit a significantly greater neurological grade t h a n SD or WKY rats (Fig. 2), indicating an increased neurological deficit ( p deficits in several additional tests o f s e n s o r y - m o t o r function after S h a m or p e r m a n e n t M C A O surgery). The results of these tests also are listed in Table 2. In the " H i n d l i m b P l a c e m e n t " test, s h a m - o p e r a t e d animals typically exhibit a n o r m a l limb placement. Following M C A O , significantly more
Perfus~on
~ Occlude MCA
100 t 50 0 I
Srain
i
FIG. 1. Example of decreased microvascular perfusion recorded/verified in core ischemic cortext area before and after permanent MCAO. Cortical perfusion after MCAO typically decreased greater than 80% of pre-occlusion baseline. The maintained/permanent decrease for 13 animals with artifact free recordings was to 15.6 + 1.7°70of baseline. Similar effects were seen in all rat strains studied.
TABLE 2 N E U R O L O G I C A L DEFICITS IN SHR
Test +
Sham (N)
Hindlimb Placement (o70 normal) 82.6 (23) Roto-Rod (sec) 126 + 19(10) Balance Beam (sec) 116 _+ 29 (5) Motor Activity** (crossings) 16 + I t o 1 5 + 1(13)
Permanent MCAO (N)
20.0 (30)* 41 _ 7 (15)* 25 _+ 5 (7)* 17 _ I to 14 + I (24)
+Testing procedures and quantitation as described in text. *Significantly different from Sham, p < 0.05. **Line crossings before to after Sham or MCAO surgery.
Permanent MCAO Neurological Grade 2.5
2.0
Grade Following Surgery
1.5
1.0
TABLE 1 M E A N A R T E R I A L PRESSURE IN C O N S C I O U S RATS
Strain
N
Mean Arterial Pressure*
SHR SD WKY
5 4 5
161.6 +_ 3.4** 123.3 _+ 6.2 121.0 _+ 4.3
0.5
0.0
*Preparation of animals and testing as described in text. **Significantly increased above SD and WKY rats; p < 0.05.
Sham
Spontaneously Hypertensive
Sprague Oawley
Wlstar Kyoto
FIG. 2. Neurological Grades in Sham (SHR: N = 8)-operated and permanent MCAO-operated rats from three strains 24 h post-surgery. Significantly larger neurological deficits were observed for SHR (N = 9) compared to normotensive SD (N = 8) and WKY (N = 7) rats. * = significantly, p < 0.05, different from Sham; + = significantly, p < 0.05, different from SD rats.
HYPERTENSION
AND ISCHEMIC SENSITIVITY
S H R failed to exhibit a n o r m a l limb placement response ( p < 0.05). Pre-test results for b o t h the "Neurological G r a d e " a n d the " H i n d l i m b P l a c e m e n t " test were always (0 _+ 0 a n d 100%0 in any group(s) o f animals tested (data not shown). F u r t h e r m o r e , p e r f o r m a n c e in the " R o t a - R o d " a n d the stationary "Balance B e a m " tests were significantly ( p < 0.05) decreased following M C A O c o m p a r e d to s h a m surgery, while the index o f m o t o r activity was not affected by M C A O compared to s h a m surgery. Brain infarct analysis. Figure 3 illustrates a typical infarct p r o d u c e d by p e r m a n e n t M C A O in S H R , as c o m p a r e d to S h a m surgery, identified 24 h after surgery following staining with T T C . These infarctions c o r r e s p o n d to volumes ranging from 128 to 179 m m 3 a n d typically were restricted to the cortex in the frontal and parietal areas. I n f a r c t i o n was not observed in S h a m (SHR) animals. Figure 4 illustrates the forebrain profile of infarction for s h a m (SHR) a n d the three strains o f M C A O - o p e r a t e d animals. No cortical infarctions were observed following sham surgery. In S H R , the infarction occupied a large p o r t i o n o f the a n t e r i o r forebrain (i.e., 20 to 4 0 % o f the n o r m a l contralateral hemisphere), but decreased in size in the more posterior f o r e b r a i n areas. A m u c h smaller profile (i.e., less t h a n 2 0 % o f the n o r m a l contralateral hemisphere) was observed in the n o r m o t e n s i v e strains, with WKY rats exhibiting the smallest infarct profile. Figure 5 illustrates the total hemispheric infarct calculated from the sum of the slice data. S H R exhibited the largest and least variable total hemispheric infarcts following p e r m a n e n t M C A O (p < 0.05). Brain swelling analysis. The swelling measure for S h a m , S H R , a n d n o r m o t e n s i v e rats was variable. For s h a m - o p e r a t e d and n o r m o t e n s i v e animals it varied a r o u n d zero t h r o u g h o u t the forebrain. Following M C A O in S H R , the size of the ipsilateral hemisphere was increased, not necessarily related to
223
Permanent MCAO Forebrain Infarction Profile 50 ¸ A. Sham Surgery ~
I'~
"E m
.
o
v
$
30
~:
2o
B. Sponlaneously Hypertensive C. Sprague Dawley
4O ~
D. Wlstar KyoIo
E
4
3
2 1 0 -1 -2 -3 -4 Distance From Bregma (ram)
-5
FIG. 4. Profile of Forebrain Hemispheric Infarction for sham (SHR; N = 8)- and permanent MCAO-operated rats from three strains (SHR; N = 9; SD; N = 8; WKY; N = 7) 24 h post-surgery. Measures were determined using image analysis/planimetry and expressed in reference to the normal contralateral hemisphere as described in methods and Fig. 3.
SHAM SURGERY
MCAO SURGERY
! a,
FIG. 3. Photographs of forebrain sections stained with triphenyltitrazolium 24 h after Sham surgery (left) or MCAO surgery (right). Eleven planar images were obtained from each side of six 2 mm thick sections of the complete forebrain and correspond approximately to I mm section surfaces from -b 5 mm to - 5 mm (i.e., from left to right, top to bottom) in reference to bregma 09). These planar images from the photographs were used in the Image Analysis System for planimetry determinations of infarct size and swelling.
224
B A R O N E E T AL. Permanent
Permanent MCAO Total Hemisheric Water Content
MCAO
Total Hemispheric I n f a r c t i o n 81.0
x
30
+ Contralateral 80.5
25
Total Percent Hemispheric
Ipsda '~'~
80.0,
20
Infarction
E EL
10
78.0,
+
77.5, Sham
Sham
Spontaneously Spragul Hypertensive Dawley
Wislar
Kyoto
FIG. 5. Total Hemispheric Infarction for sham (SHR; N = 9) and permanent MCAO-operated rats from three strains 24 h post-surgery. Measures determined as described in methods and Fig. 3. Significantly larger forebrain infarctions were observed for SHR (N = 9) compared to SD (N = 8) and WKY (N = 7) rats. * =significantly, p < 0.05, different from Sham; + = significantly, p < 0.05, different from SD rats.
the degree of infarction but primarily in the more a n t e r i o r a n d more posterior aspects of the forebrain. The total hemispheric swelling calculated from the sum of the slice data is illustrated in Fig. 6 S H R exhibited a significant increase in hemispheric size that was not o b t a i n e d in S h a m - o p e r a t e d S H R or n o r m o tensive animals ( p < 0.05). Figure 7 illustrates the significant
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FIG. 6. Total Hemispheric Swelling for sham (SHR)- and permanent MCAO-operated rats from three strains 24 h post-surgery. Percent change in ipsilateral hemispheric size was determined as described in the methods and Fig. 3. A significant increase in ipsilateral hemispheric size was observed in SHR MCAO-surgery animals (N = 9) that was not observed in sham (N = 8), SD (N = 8) or WKY (N = 7) rats. * = significantly, p < 0.05, different from Sham.
MCAO
Surgery FIG. 7. Percent water content for ipsilateral and contralateral hemispheres in sham (N = 8)- and MCAO (N = 9)-operated SHR 24 h post-surgery. A significant increase (*; p < 0.01) in the ipsilateral hemispheric water content (i.e., edema) occurred following MCAO but not following sham surgery.
increase in ipsilateral hemispheric water content that occurs 24 h after M C A O (p < 0.01) in SHR. No change in hemispheric water content was produced by sham surgery.
Temporary MCA 0 with Reperfusion (24 h) in SHR Cortical microvascular perfusion. Initial studies were carried out to characterize the effects of M C A O with reperfusion in SHR in order to select the a p p r o p r i a t e occlusion-time parameter for c o m p a r i s o n to n o r m o t e n s i v e animals. As for perm a n e n t M C A O , a similar decrease in local microvascular perfusion was verified in the primary ischemic cortical area o f S H R during t e m p o r a r y M C A O . Figure 8 illustrates these results for several occlusion times that were studied. During M C A O , cortex perfusion was decreased persistently for the total d u r a t i o n of the controlled occlusion. Also, u p o n reperfusion regional cortical blood flow recovered to pre-occlusion levels. In some animals cortical blood flow was recorded for periods of up to 60 rain post-reperfusion a n d cortical perfusion was observed to remain at p r e - M C A O levels. A similar pattern of cortical blood flow changes in the primary ischemic area (i.e., a decrease followed by a complete recovery o f perfusion) was observed in n o r m o t e n s i v e animals during M C A O with reperfusion (data not shown). Neurological deficits. Figure 9 illustrates Neurological G r a d e results for t e m p o r a r y M C A O of 0 min (sham surgery) to 300 min followed by 24 h reperfusion a n d for p e r m a n e n t (24 h) M C A O in SHR. The Neurological G r a d e was very sensitive to even short periods of t e m p o r a r y M C A O . A n occlusion-time-related increase in neurological deficit was observed (p < 0.05). Occlusion-times of 40 rain and a b o v e significantly increased the grade, with m a x i m u m grades (i.e. similar to that which occurs following p e r m a n e n t M C A O ) achieved after 160 min o f t e m p o r a r y M C A O . Neuropathological changes. Figure 10 illustrates the forebrain profile o f infarction for several periods o f t e m p o r a r y occlusion. A clear ischemia-time-related increase in the forebrain infarct profile was observed, with the largest infarct profile observed following p e r m a n e n t M C A O . Figure 11 illus-
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