Diffusion- and T2-weighted imaging: Evaluation of oedema reduction in focal cerebral ischaemia by the calcium and serotonin antagonist levemopamil

MagneIic Resonance Printed in the USA.

II, pp.401-409. 1993

Imaging, Vol. All rights reservcd.

Copyright

0

0730-725X/93 56.00 + .oO 1993 Pergamon Press Ltd.

0 Original Contribution DIFFUSION- AND &WEIGHTED IMAGING: EVALUATION OF OEDEMA REDUCTION IN FOCAL CEREBRAL ISCHAEMIA BY THE CALCIUM AND SEROTONIN ANTAGONIST LEVEMOPAMIL J~RGEN SEEGA AND BERND ELGER Research and Development, Molecular Pharmacology and Screening, Knoll AG, Knollstr. 50, D-6700 Ludwigshafen, Germany Magnetic resonance imaging (MRI) was used to evaluate beneficial drug effects in a rat model of focal cerebral ischaemia. Extent of cerebral oedema was measured on T2-weighted images 24 hr after permanent middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. Areas of increased signal intensity strongly correlate with histochemlcally determined areas of ischaemia in corresponding brain planes (r = 0.84; p < .OOl). In a separate cohort of animals, spatial progression of oedema formation was studied at 3, 24,48, and 72 hr after MCAO showing a maximum extent at 48 hr. Early events in cerebral ischaemia were monitored using diffusionweighted imaging. Effects of levemopamil [formerly (S)-emopamiIl, a calcium and serotonin antagonist, and the reference compound isradipine were quantified on high resolution T2-weighted spin-echo images 24 hr after MCAO. Combined pre- and posttreatment with isradipine showed a 21% inhibition of oedema progression. Application of a single dose (10 mg/kg) of levemopamil either 30 min before or 2 hr after MCAO revealed a diminution of oedematous areas by 19% and 2S70, respectively. Levemopamil reduces the extent of ischaemic brain oedema in an established stroke model. Keywords: Diffusion weighted imaging; MRI of brain oedema; Cerebral ischaemia; Stroke; Serotonin receptor antagonist.

INTRODUCTION

rotonin antagonist levemopamil [recommended INN by the WHO for (S)-emopamil]. Spontaneously hypertensive rats (SHR) were chosen because this strain displays the highest reproducibility of infarct topography and volume and is, therefore, especially suited for routine use in studies on pathogenesis and treatment of ischaemic brain injury.5 Animals were treated either 30 min prior to or 2 hr following MCAO with a single injection of levemopamil. Both protocols may be clinically relevant. Prophylactic treatment may be indicated as preoperative care, for example, in the case of surgery of the heart or carotid artery. Posttreatment may be beneficial in the acute clinical situation of stroke. Ischaemic lesions were quantified from T2-weighted spin-echo images taken 24 hr after MCAO. The technique of diffusion-weighted imaging has proven to be suitable to study the formation of the ischaemic lesions in the early phase of acute stroke.6 Because of the noninvasive character of MRI, the combination of both the diffusion-weighted and the T2-weighted imaging tech-

In focal cerebral ischaemia, the development of infarcts is associated with the formation of severe oedema within 24 hr after permanent occlusion of intracranial arteries such as the middle cerebral artery. The oedematous brain swelling causes increased tissue pressure leading to a compression of the microvasculature,’ which further aggravates the impact of the primary ischaemia. In consequence, inhibition of oedema progression by effective drug treatment may restrict the final infarct and thereby ameliorate the clinical outcome of stroke patients. It has been demonstrated that motor deficits and memory disturbance closely correlate with brain oedema.2 Magnetic resonance imaging (MRI) has frequently been applied in the clinical diagnosis of brain lesions in the territory of the middle cerebral artery.3,4 The aim of our MRI study was to determine the extent of brain oedema 24 hr after middle cerebral artery occlusion (MCAO) in rats treated with the calcium and seRECENED

g/24/92;

Address

ACCEPTED 11/17/92. 401

correspondence

to Dr Jiirgen

Seega.

402

Magnetic Resonance Imaging 0 Volume 11, Number 3, 1993

niques allow the progression of lesion formation to be monitored intraindividually. As a reference compound, isradipine was tested to validate the methodological approach for the demonstration of beneficial drug effects. The cerebroprotective potential of this calcium channel blocker is superior to other dihydropyridines, for example, nimodipine, nitrendipine, or nicardipine,7 as has been demonstrated by recent MR studies in the same animal model. Levemopamil, which is a calcium channel blocker of the phenylalkylamine type and a serotonin-$-receptor antagonist in addition, has already shown remarkable neuronal protection in numerous studies on global and focal cerebral ischaemia. *-” MATERIALS AND METHODS Animals and Surgery Male spontaneously hypertensive rats (250-280 g) were maintained under standard conditions (12 hr day/ night cycle, 22”C, 50% humidity) with free access to food and tap water before and after surgery. Occlusion of the left proximal middle cerebral artery was performed by the method of Tamura et al.i3 in animals anaesthetized with sodium pentobarbital (60 mg/kg IP). A vertical 2 cm skin incision was made between the left eye and the left ear. The temporalis muscle was divided and retracted in order to expose the zygoma and the squamosal bone. Parts of the temporalis muscle and the zygoma were removed. The left MCA was exposed through a burr hole craniectomy (2 mm diameter) drilled in close proximity to the foramen ovale. The stem of the MCA was electrocauterized with bipolar forceps. The temporalis muscle was put back into position and the skin was sutured. Rectal temperature was maintained at 37°C with a thermostatically regulated heating pad. Drugs Levemopamil [(2S)-2-isopropyl-5-(methylphenethylamino)-2-phenylvaleronitrile hydrochloride] was given in distilled water as a single IP injection (10 mg/ kg) either 30 min before MCAO in the pretreatment group or 2 hr after MCAO in the posttreatment group. Isradipine (Lomir@) was dissolved in ethanol/PEG 400 and subcutaneously injected (0.1 mg/kg) at 45 min before and again 45 min and 135 min after MCAO according to Sauter and Rudin.14 Animals in the two independent control groups received the corresponding vehicle solutions. In addition to the control and the treatment groups two animals were sham operated without further treatment.

MR Imaging MRI experiments were carried out in vivo on a General Electric CSI-II 2.0 T NMR system equipped with Acustar” self-shielded gradient coils (maximum gradient strength + 20 G/cm, 15 cm clear bore). ‘H-MR imaging was performed using a home-built low pass birdcage resonator (inner diameter: 60 mm) consisting of eight segments separated by 22 pF ATC chip capacitors (modified from Hayes et al.“). The proton frequency was tuned to 85.5 MHz and the water signal of the head was shimmed to a proton line width of typically 0.6 ppm. For the study of oedema formation MR images were measured in a multislice mode at 3,24,48, and 72 hr following MCAO in four adjacent coronal brain planes (slice thickness: 2 mm) located 3,6,9 and 12 mm anterior to the interaural line. The stereotactic coordinates are referred to the atlas of Paxinos and Watson.16 The technique of diffusion-weighted imaging is required to visualize the early events of cerebrovascular disorders.i7*i8 Therefore, multislice diffusionweighted images (TR = 1.8 set, TE = 80 msec, NA = 8) with diffusion gradients of 20 msec duration and an amplitude of 5.5 G/cm were taken at 3 hr after MCAO. At the later time-points, multislice Tz-weighted spinecho images (TR = 2.0 set; TE = 70 msec) were obtained with a field of view of 50 mm in which four scans were averaged for each one of the 128 phase-encoding steps, resulting in a total acquisition time of 17 min. The effects of levemopamil and isradipine were detected 24 hr after MCAO in T&weighted images (TR = 1.5 set, TE = 80 msec) of transversal brain planes located + 4.5 mm and + 5.5 mm dorsal to the interaural line, respectively. The field of view used yielded an in plane resolution of 195 pm providing a reliable differentiation between normal and injured brain tissue. Animals were placed in a home-built Plexiglas stereotactic holder fitting into the birdcage resonator. Image processing and analysis were performed on a SpecStation’ (SUN 4/l 10) using the MRIDMAGE” software (New Methods Research, Inc.; release 1.5). Total brain planes and lesioned regions were quantified, respectively, using a segmentation function which automatically determines a threshold. Based on these thresholds an automatic contour setting is performed, leading to the voxel sum in the respective regions of interest. Region of interest (ROI) image analyses were performed in the ischaemic parietal cortex and the corresponding nonischaemic contralateral region. A signal intensity ratio (SIR) has been defined as the ROI image intensity ratio of an abnormal, ischaemic region over that of the normal, contralateral side in the T2and diffusion-weighted MRI experiments.6

Levemopaml

for edema reduction in focal cerebral ischaemia 0 J. SEEGA AND B. ELCER

Histochemistry For histochemical quantification of the cerebral ischaemia, the method of Bederson et aI.19was slightly modified. Rats were sacrificed by an intracardiac injection (0.2 ml/animal) of T61 (Hoechst) immediately after the MR measurements. Coronal slices of rapidly removed brains were incubated in 1% TTC (2,3,5-triphenyltetrazolium chloride) in phosphate-buffered saline at 37°C for 45 min and then stored in 4% buffered Formalin for at least 6 hr. The ischaemic areas were quantified by a computerized image analysis system (Kontron CARD10 200) on scaled photographs (Kodak Ektachrome 50) of the posterior surfaces of the fixed brain sections. Statistical Analysis All results are presented as mean + SEM. Differences of means were analyzed using Dunnett’s test.20 Differences were considered significant at p < .05. RESULTS Temporal and Spatial Development of Ischaemic Lesion Diffusion-weighted images using strong gradients (5.5 G/cm) display a significant hyperintensity in the ischaemic region as early as 45 min after the onset of ischaemia whereas T,-weighted spin-echo images fail to show the brain injury up to 5-6 hr postocclusion. In our studies, massive brain injury was already observed in coronal diffusion-weighted images within 3 hr after MCAO (Fig. 1). At this early time point, diffusion-weighted images displayed a SIR of 1.63 f 0.13 in the parietal cortex. For comparison, the SIR of the same brain region was 1.78 f 0.07 in Tz-weighted images after 24 hr. The development of ischaemic lesion was followed in corresponding brain planes by Tz-weighted MR imaging for 3 days after MCAO (Fig. 2, Table 1). After 24 hr, the lesion showed only a minor increase in the central portion of the infarction (plane 3), whereas a 1Zfold increase in lesion size was observed in posterior brain regions (plane 1). A further augmentation of brain lesion by 23% and 29% was measured 48 hr after MCAO in the posterior brain planes 1 and 2, respectively. Altogether, a maximum extent of the lesion was found after 48 hr. Volumes of lesioned regions declined at 72 hr after MCAO. Correlation of the Extent of Brain Lesion Measured by MRI and Histochemistry Twenty-four hours after MCAO, the extent of cerebral injury was determined in four coronal brain planes of control rats by Tz-weighted MR imaging. Immediately after the noninvasive measurements, the

403

Table 1. Temporal and spatial development of ischaemic lesion after MCAO in spontaneously hypertensive rats assessed by MR imaging in four coronal brain planes Time of MRI Brain plane 1(3 mm) 2 (6 mm) 3 (9 mm) 4 (12 mm)

3 hr

24 hr

48 hr

OS? 0.4 6.5 f 2.1 9.1 f 2.4 8.0 + 1.7 17.8 -+ 2.3 23.2 z!z 1.0 28.6 f 1.1 32.2 f 1.5 37.2 f 1.4 23.2 + 2.8 30.7 f 1.4 31.2 f 1.4

72

hr

7.5 k 2.3 20.8 -+ 1.3 33.3 f 1.1 27.9 f 2.3

n = 6. The four coronal brain planes are located 3,6,9 and 12 mm anterior to the interaural line. Values represent relative areas of lesion (% of total brain area), mean f SEM.

animals were sacrificed for the histochemical quantification (TIC-staining) of the brain lesion. Comparison of the areas of increased signal intensity on MR images with the absence of vital tissue staining in corresponding tissue slices revealed a strong correlation (r = 0.84; p < .OOl) as shown in Fig. 3. Response of Ischaemic Brain Lesion to Levemopamil Treatment In order to determine the effect of levemopamil on ischaemic brain lesion T2-weighted transversal spinecho images were taken 24 hr after permanent occlusion of the left middle cerebral artery in SHRs. Well demarcated areas of enhanced signal intensity were visible in the ipsilateral hemispheres of the saline and levemopamil-treated groups, whereas images of untreated sham operated animals showed no differences between the left and the right hemispheres. Both cortical and subcortical regions were affected by ischaemia in MCAoccluded rats (Figs. 4A, 4C). Treatment with levemopamil(l0 mg/kg IP) either 30 min before or 2 hr after MCAO caused a diminution of the ischaemic brain lesion. Pretreatment with levemopamil (n = 9) resulted in a 19% reduction of the injured regions, (calculated as percentage lesion of total brain in the + 4.5 mm stereotactic plane) compared with control (Table 2). In the same stereotactic plane a more pronounced protective effect (25% lesion reduction) was observed in the group with the 2 hr delayed levemopamil treatment (n = 9). In both levemopamil groups the reduction of the lesion was significantly different (p < .05) compared to the salinetreated controls (n = 9). A further series of experiments was performed in order to study the effects of the reference compound isradipine on ischaemic brain injury. Animals were treated either with 0.1 mg/kg SC isradipine (n = 10)

404

Magnetic Resonance Imaging 0 Volume 11, Number 3, 1993

Fig. 1. Diffusion-weighted spin-echo images (TR = 1.8 set, TE = 80 msec, NA = 8) of the rat head taken in coronal brain planes 3,6,9 and 12 mm anterior to the interaural tine 3 hr after middle cerebral artery occlusion (MCAO). Note the increased signal intensity in the ischaemic region of the left hemisphere. A water phantom was positioned next to the head.

or with the vehicle solution (ethanol/PEG 400; n = 10) at 45 min prior to and again at 45 and 135 min following occlusion of the middle cerebral artery. In isradipine-treated animals the mean injured region in the brain plane under investigation (+5.5 mm dorsal to the interaural line) was only 12.7 f 0.7% (o/olesion of total brain area), whereas in the control group the lesion

extent was 16.1 f 0.4% (Table 3). Thus, combined preand posttreatment with isradipine caused a 21% reduction of ischaemic brain lesion compared to the vehicletreated controls (p < .Ol). The reduction was most pronounced in posterior cortical brain regions of spontaneously hypertensive rats treated with levemopamil or isradipine (Figs. 4B, 4D).

Levemopamil for edema reduction in focal cerebral ischaemia l J.

SEEGAAND B. ELGER

405

Fig. 2. 7”-weighted spin-echo images (TR = 2.0 set, TE = 70 msec, NA = 4) taken from the same rat 24 hr after middle cebrain planes as in Fig. 1.

rebral artery occlusion (MCAO) in corresponding

DISCUSSION This study produced two clear results: MRI and histological estimates of lesion size agreed quantitatively, and levemopamil reduced lesion size by 25% when given as late as 2 hr after lesion onset. MR imaging is applied in clinical diagnosis of cerebral diseases in the same way as we use it for pharmacolog-

ical research. Numerous studies have demonstrated that the regions of lesion observed on MR images correspond to infarcted areas determined postmortem by histological techniques, both in human stroke and various animal models.2’-26 A remarkable conformity of lesioned regions has repeatedly been reported for T2-weighted MR images and postmortem vital tissue staining (TTC).27-29 However, most descriptions of

Levemopamlfor edemareductionin focal cerebral ischaemia 0 J.

SEEGA AND B. ELGER

407

Fig. 4. T.-weighted (2000/70) spin-echo brain images of control (a,c) and isradipine-treated (b,d) rats 24 hr after permanent middle cerebral artery occlusion (MCAO). Reduction of ischaemic lesion in the posterior region (arrow) is demonstrated in transversal (a, b) and coronal (c,d) brain planes.

area our results indicate a progressive extension predominantly from anterior to posterior cortical brain regions in focal cerebral ischaemia. After 72 hr, a decrease was observed in the size of areas with increased signal intensity in T&weighted images. This finding, together with the recently described correlation of tissue water content and size of lesion in T&weighted

images,34 indicates that MR imaging is capable of visualizing cerebral oedema which is a clinically significant phenomenon occurring in stroke. Two independent studies were performed to demonstrate the effect of the calcium antagonist isradipine and the calcium and serotonin antagonist levemopamil on brain oedema, respectively. Prior to the MRI study

408

Magnetic Resonance Imaging 0 Yolume 11, Number 3, 1993

of the cerebroprotective potential of levemopamil the experimental protocol, which was slightly modified with respect to Sauter and Rudin,14 was validated using the reference compound isradipine. MRI measurements were performed 24 hr after MCAO because this is a frequently used time to monitor pathological tissue changes in models of cerebral ischaemia.5s7z’9,34*35 Interestingly, oedema reduction by both compounds, levemopamil and isradipine, was most pronounced in the posterior brain regions (temporal and occipital cortex) which displayed little or no signal intensity change in early diffusion-weighted MR images. Accordingly, brain oedema and infarct reductions by these drugs may be attributed to beneficial effects in the penumbra of the infarcted region. Levemopamil acts preferentially on cerebral blood vessels. 36It displays a high affinity to the 5-HTz-receptor and thereby may diminish vasospasms caused by increased levels of serotonin during ischaemia. In that way the oxygen supply in the periphery of the ischaemic lesion may be improved and the progression of ischaemic lesions inhibited. Reduction of brain oedema in various animal studies suggests that the clinical benefits of successful therapeutic oedema reduction may be corresponding improvements in the neurological scores2*14and decreased mortality.37 Significant oedema reduction by levemopamil in the SHR is not only achieved by pretreatment but also when the substance is given as late as 2 hr after the onset of focal cerebral ischaemia. In both cases, the administration of a single injection of levemopamil yielded beneficial effects comparable to the results of combined pre- and posttreatment in isradipine-treated hypertensive rats. Similarly, infarct reductions have been observed in normotensive rats when levemopamil administration starts 1 or 2 hr after MCAO.“*” Treatment initiated 3 hr postocclusion remained, however, without effect. Thus, the postischaemic therapeutic window for cerebroprotection by levemopamil may be only up to 2 hr after MCAO in the rat, although oedema extent still increases substantially between 3 and 48 hr after the insult. This may be attributed to changing pathophysiological conditions during the development of brain oedema. During the first hours of oedema formation (cytotoxic oedema), blood-brain barrier function for macromolecules is still intact.35,38 In the advanced stage of oedema formation (vasogenic oedema), bloodbrain barrier function is impaired and brain protection is apparently rendered more difficult. Hence, early hospital admission of patients is required for successful therapeutic intervention. At present, only a small number of stroke patients are amenable to treatment in the hospital within less than 6 hr after the onset of deficit, except for a few stroke centres with special conditions.

However, this number could be considerably increased by improved transportation and public education, as has been suggested repeatedly.39*40 REFERENCES 1. Klatzo, I.; Seida, M.; Wagner, H.G.; Ting, P. Features of ischemic brain edema. In: Krieglstein, J. (Ed). Pharmacology of Cerebral Ischemia. Amsterdam: Elsevier Science Publishers; 1986: pp. 23-30. 2, Tominaga, T.; Ohnishi, S.T. Interrelationship of brain edema, motor deficits, and memory impairment in rats exposed to focal ischemia. Stroke 20:513-518; 1989. 3. Lechner, H.; Schmidt, R.; Bertha, G.; Justich, E.; Offenbather, H.; Schneider, G. Nuclear magnetic resonance image white matter lesions and risk factors for stroke in normal individuals. Stroke 19:263-265; 1988. 4. Steinbrich, W.; Friedmann, G.; Pawlik, G.; BiicherSchwarz, H.G.; Heiss, W.D. MR bei ischlmischen Hirnerkrankungen. Ein Vergleich mit CT, PET (‘*Fluordeoxyglukose) und angiographischen Ergebnissen. Fortschr. Riintgenstr. 145:173-181; 1986. 5. Brint, S.; Jacewicz, M.; Kiessling, M.; Tanabe, J.; Pulsinelli, W. Focal brain ischemia in the rat: Methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J. Cereb. BloodFlow Metab. 8:474-485; 1988. 6. Moseley, M.E.; Cohen, Y.; Mintorovitch, J.; et al. Early detection of regional cerebral ischemia in cats: Comparison of diffusion- and T,-weighted MRI and spectroscopy. Magn. Reson. Med. 14:330-346; 1990. 7. Sauter, A.; Rudin, M.; Wiederhold, K.H.; Hof, R.P. Cerebrovascular, biochemical, and cytoprotective effects of isradipine in laboratory animals. Am, J. Med. 86(Suppl. 4A): 134-146; 1989. 8. Block, F.; Jaspers, R.M.A.; Heim, C.; Sontag, K.-H. S-emopamil ameliorates ischemic brain damage in rats: Histological and behavioural approaches. Life SC;. 47: 1511-1518; 1990. 9. Lin, B.; Dietrich, W.D.; Busto, R.; Ginsberg, M.D. (S)emopamil protects against global ischemic brain injury in rats. Stroke 21:1734-1739; 1990. 10. Morikawa, E.; Ginsberg, M.D.; Dietrich, W.D.; Duncan, R.C.; Busto, R. Postischemic (S)-emopamil therapy ameliorates focal ischemic brain injury in rats. Stroke 22:355-360; 1991. 11. Nakayama, H.; Ginsberg, M.D.; Dietrich, W.D. (S)-emopamil, a novel calcium channel blocker and serotonin SZ antagonist, markedly reduces infarct size following middle cerebral artery occlusion in the rat. Neurology 38: 1667-1673; 1988. 12. Szabo, L.; Hofmann, H.P. (S)-emopamil, a novel calcium and serotonin antagonist for the treatment of cerebrovascular disorders. 3rd communication: Effect on postischemic cerebral blood flow and metabolism, and ischemic neuronal cell death. Drug Res. 39:314-319; 1989.

Levemopamil for edema reduction in focal cerebral ischaemia 0 J. 13. Tamura, A.; Graham, D.I.; McCulloch, J.; Teasdale, G.M. Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. J. Cereb. Blood Flow Metab. 1:53-60; 1981. 14. Sauter, A.; Rudin, M. Calcium antagonists reduce the extent of infarction in rat middle cerebral artery occlusion model as determined by quantitative magnetic resonance imaging. Stroke 17:1228-1234; 1986. 15. Hayes, C.E.; Edelstein, W.A.; Schenck, J.F.; Mueller, O.M.; Eash, M. An efficient, highly homogeneous radiofrequency coil for whole-body NMR imaging at 1.5 T. J. Magn. Reson. 63:622-628; 1985. 16. Paxinos, G.; Watson, C. The Rat Brain in Stereotactic Coordinates. Sydney, Australia: Academic Press; 1982. 17. Le Bihan, D.; Breton, E.; Lallemand, D.; Grenier, P.; Cabanis, E.; Laval-Jeantet, M. MR imaging of intravoxel incoherent motions: Application to diffusion and perfusion in neurologic disorders. Radiology 161:401-407; 1986. 18. Le Bihan, D.; Breton, E.; Lallemand, D.; Aubin, M.-L.; Vignaud, J.; Laval-Jeantet, M. Separation of diffusion and perfusion in intravoxel incoherent motion MR imaging. Radiology 168:497-505; 1988. 19. Bederson, J.B.; Pitts, L.H.; Germane, S.M.; Nishimura, M.C.; Davis, R.L.; Bartkowski, H.M. Evaluation of 2,3,5triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17:1304-1308; 1986. 20. Dunnett, C.W. A multiple comparison procedure for comparing several treatments with a control. J. Am. Statistical Association 50:1096-l 121; 1955. 21. Buonanno, F.S.; Pykett, I.L.; Kistler, J.P.; et al. Cranial anatomy and detection of ischemic stroke in the cat by nuclear magnetic resonance imaging. Radiology 143: 187-193;

AJNR 7:7-11; 1986. 29. Kucharczyk, J.; Chew, W.; Derugin, N.; et al. Nicardi-

pine reduces ischemic brain injury. Magnetic resonance imaging/spectroscopy study in cats. Stroke 20:268-274; 1989. 30. Barone, EC.; Clark, R.K.; Feuerstein,

G.; Lenkinski, R.E.; Sarkar, S.K. Quantitative comparison of magnetic resonance imaging (MRI) and histologic analyses of focal ischemic damage in the rat. Bruin Res. BUN. 26:285-

291; 1991. 3 1. Allegrini, P.R.; Sauer, D.; Pataki, A.; Cosenti, A.; Fagg,

32.

33.

34.

35.

22. Dewitt,

274; 1985. 24. Pykett, I.L.; Buonanno,

ES.; Brady, T. J.; Kistler, J.P. True three-dimensional nuclear magnetic neuro-imaging in ischemic stroke: Correlation of NMR, X-ray CT and pathology. Stroke 14:173-177; 1983. 25. Sauter, A.; Rudin, M.; Wiederhold, K.H. Reduction of neural damage in irreversible cerebral ischemia by calcium antagonists. Neurochem. Pathol. 9:21 l-236; 1988. 26. Zabramski, J.M.; Spetzler, R.F.; Kaufman, B. Magnetic resonance imaging: Comparative study of radiofrequency pulse techniques in the evaluation of focal cerebral ischemia. Neurosurgery 16:502-510; 1985. 27. Bose, B.; Jones, S.C. ; Lorig, R.; Friel, H.T.; Weinstein,

409

M.; Little, J.R. Evolving focal cerebral ischemia in cats: spatial correlation of nuclear magnetic resonance imaging, cerebral blood flow, tetrazolium staining and histopathology. Stroke 19:28-37; 1988. 28. Brant-Zawadzki, M.; Pereira, B.; Weinstein, P.; et al. MR imaging of acute experimental ischemia in cats.

1982.

L.D.; Kistler, J.P.; Miller, D.C.; Richardson, E.P.; Buonanno, ES. NMR-neuropathologic correlation in stroke. Stroke 18:342-351; 1987. 23. Kato, H.; Kogure, K.; Ohtomo, H.; et al. Correlations between proton nuclear magnetic resonance imaging and retrospective histochemical images in experimental cerebral infarction. J. Cereb. Blood Flow Metab. 5:267-

SEEGA AND B. ELGER

36.

G.E. Determination of infarction size after middle cerebral artery occlusion in rat by MR imaging: Correlation with histological method (abstr). In: Book of Abstracts: Society of Magnetic Resonance in Medicine 1991. Berkeley, CA: Society of Magnetic Resonance in Medicine; 1991: 941. Elger, M.; Bankir, L.; Kriz, W. Morphometric analysis of kidney hypertrophy in rat after chronic potassium depletion. Am. J. Physiol. 262:F656-F667; 1992. Moseley, M.E.; Kucharczyk, J.; Mintorovitch, J.; et al. Diffusion-weighted MR imaging of acute stroke: Correlation with 7”-weighted and magnetic susceptibilityenhanced MR imaging in cats. AJNR 11:423-429; 1990. Naruse, S.; Aoki, Y.; Takei, R.; Horikawa, Y.; Ueda, S. Effects of atria1 natriuretic peptide on ischemic brain edema in rats evaluated by proton magnetic resonance method. Stroke 22:61-65; 1991. Ishimaru, S.; Hossmann, K.-A. Relationship between cerebral blood flow and blood-brain barrier permeability of sodium and albumin in cerebral infarcts of rats. Acta Neurochirur. Suppl. 51:216-219; 1990. Hofmann, H.P.; Raschack, M.; Unger, L. (S)-emopamil, a novel calcium and serotonin antagonist for the treatment of cerebrovascular disorders. 1st communication: Pharmacological profile. Arzneim-ForschIDrug

Res. 39:304-308; 1989. 37. Harrison, M. J.G.; Brownbill, D.; Lewis, P.D.; Russel,

R. Cerebral edema following carotid artery ligation in the gerbil. Arch. Neurol. 28:389-391; 1973. 38. Menzies, S.A.; Hoff, J.T.; Betz, A.L. Extravasation of albumin in ischaemic brain oedema. Acta Neurochirur. Suppl. 51:220-222; 1990. 39. Herdersches, D.; Limburg, M.; Hijdra, A.; Bollen, A.; Pluvier, J.; teWater, W. Timing of hospital admission in a prospective series of stroke patients. Cerebrovusc. Dis. 1:165-167; 1991. 40. Harper, G.D.; Haigh, R.A.; Potter, J.F.; Castleden, C.M. Factors delaying hospital admission after stroke in Leicestershire. Stroke 23:835-838; 1992.