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Oedema reduction by levemopamil in focal cerebral ischaemia of spontaneously hypertensive rats studied by magnetic resonance imaging
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European Journal of Pharmacology254 (1994) 65-71
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Oedema reduction by levemopamil in focal cerebral ischaemia of spontaneously hypertensive rats studied by magnetic resonance imaging Bernd Elger *, Jiirgen Seega, Manfred Raschack Research and Development, Knoll AG, P.O. Box 21 08 05, D-67008 Ludwigshafen, Germany
(Received 12 July 1993;revised MS received 6 December 1993; accepted 10 December 1993)
Abstract The effect of treatment with the C a 2+ channel blocker and 5-HT2 receptor antagonist levemopamil (recommended INN for (S)-emopamil) on the extent of ischaemic brain oedema was studied by magnetic resonance imaging in vivo. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent middle cerebral artery occlusion. The treatment consisted of slow intravenous injections of an aqueous solution of levemopamil given immediately after middle cerebral artery occlusion and again 2 h and 4 h later. One group of animals (n = 17) received 3 × 2 mg/kg of levemopamil (total dose: 6 mg/kg) and another group (n = 13) received 3 × 4 mg/kg (total dose: 12 mg/kg). Saline was administered to the controls (n = 16) at corresponding times. High-resolution T2-weighted spin echo images were obtained 24 h after middle cerebral artery occlusion from two transversal brain planes (4.5 mm and 6.5 mm dorsal to the interaural line). Dose-dependent reductions of brain oedema were achieved in both brain planes. The lower dose of levemopamil reduced the extent of oedema significantly (P < 0.05) by 20 + 3.7% in the upper and by 21 + 3.8% in the lower brain plane as compared to the controls (means + S.E.M.). The higher dose diminished the extent of oedema in the same planes by 30 + 3.5% and 31 + 4.0%, respectively. Dose-dependent reductions of infarct size, as determined by vital tissue staining using 2,3,5-triphenyltetrazolium chloride (TTC), were observed in the levemopamil-treated groups. Body temperature was not affected by levemopamil, suggesting direct cerebroprotection by this drug. The results of this magnetic resonance imaging study indicate that the extent of brain oedema and infarction is dose dependently reduced by treatment with levemopamil in a clinically relevant stroke model. Key words: Magnetic resonance imaging; NMR tomography; Brain edema; Stroke; 5-HT receptor antagonist; Ca 2+ channel
blocker
I. Introduction Clinically, the formation of oedema in cerebral disorders such as stroke and trauma is a major complicating factor which causes aggravation of the diseases (Goldstein and Davis, 1990). An increased tissue pressure in oedematous areas may reduce blood flow in the adjacent tissues and thus extend the ischaemically affected territory (Klatzo et al., 1986). Furthermore, oedema may influence the neuronal control of vegetative function by compression in areas remote from the lesion and thereby cause the death of patients (Shaw et al., 1959). Evidence is accumulating that serotonin is
* Corresponding author. Tel. (0621) 589-2802, fax (0621) 589-2050. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(93)E0887-X
involved in the formation of ischaemic cerebral oedema, as has recently been reviewed by Kumami et al. (1990). During brain ischaemia there is a marked increase in extracellular serotonin levels (Phebus and Clemens, 1989). Thus, the aim of the present study was to investigate the effects of levemopamil (recommended INN for (S)-emopamil) on the extent of oedema in focal cerebral ischaemia. This agent is a stereoselective antagonist of the cerebral 5-HT 2 receptor, having a g i of 4.4 n m o l / l for ketanserin displacement (Raschack et al., 1989). Other features are L-type Ca2+-channel antagonism, high cerebral availability and lower cardiovascular activity than e.g. the Ca 2÷ channel blocker verapamil. Middle cerebral artery occlusion in rats was used as a widely accepted clinically relevant animal model for human stroke (Petito, 1986; Siesj6, 1986;
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Ginsberg and Busto, 1989). However, in contrast to earlier studies on the beneficial effects of levemopamil in this model (Nakayama et al., 1988; Morikawa et al., 1991), we performed the experiments with spontaneously hypertensive rats (SHR) instead of normotensive rats for two reasons. Firstly, infarct variability is much lower in SHR than in normotensive rats (Duverger and MacKenzie, 1988). Secondly, stroke frequently occurs in hypertensive patients (Dunbabin and Sandercock, 1990). Since significant infarct reduction may be found only in normotensive rats but not in SHR, as has been shown for the N-methyl-D-aspartate antagonist MK-801 (Roussel et al., 1992), positive results obtained with SHR may aid in the initiation of clinical drug testing. The influence of intravenous treatment with levemopamil on brain oedema was measured by magnetic resonance imaging in vivo. Magnetic resonance imaging is a widely used technique for medical diagnosis and, furthermore, it is well suited to determine drug effects on ischaemic brain oedema (Kucharczyk et al., 1989; Naruse et al., 1991; Allegrini and Sauer, 1992; Sauer et al., 1993). T2-weighted spin echo imaging was applied to display the spatial extent of brain oedema because a high correlation between tissue water content and oedematous area on T 2weighted images has been demonstrated (Naruse et al., 1991). With regard to clinical stroke therapy, the intravenous route was chosen in the present study and different doses of levemopamil were tested.
2. Materials and methods
Our experiments were performed in accordance to the requirements of the German laws for the protection of animals (Deutsches Tierschutzgesetz).
2.1. Animals Male SHR (280-300 g) were obtained from Mollegaards (Skensved, Denmark) and maintained under standard conditions (12 h day/night cycle, 22°C, 50% humidity) with free access to food and tap water before and after surgery. Systolic arterial blood pressure of conscious animals was measured by tail-cuff plethysmography using a piezo crystal a few days before surgery as described elsewhere (Gerold and Tschirky, 1968). Rectal temperature was determined with a thermoelectrical probe.
2.2. Surgery The animals were anaesthetized with sodium pentobarbital (60 mg/kg i.p.) and placed on a thermostatically regulated heating pad to maintain rectal temperature at 37°C. The permanent occlusion of the left
proximal middle cerebral artery was performed by the method of Tamura et al. (1981).
2.3. Magnetic resonance imaging The animals were anaesthetized with sodium pentobarbital (60 mg/kg i.p.) for magnetic resonance imaging 24 h after middle cerebral artery occlusion. Experiments were carried out in vivo with a General Electric CSI-II 2.0 Tesla NMR system equipped with Acustar self-shielded gradient coils (maximum gradient strength + 20 gauss/cm, 15 cm clear bore). Magnetic resonance imaging was performed by 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., 1985). The animals were placed in a home-built plexiglas stereotactic holder fitting into the birdcage resonator. 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. T2-weighted images (TR = 2 s, TE = 80 ms, NA = 4) were taken of transversal brain planes located +4.5 mm and + 6.5 mm dorsal to the interaural line. The stereotactic coordinates are from the atlas of Paxinos and Watson (1982). Further measurements dorsal and ventral to these two adjacent planes were not performed to avoid erroneous overestimation of oedema due to partial volume effects. Images displayed 2-mm thick slices with an in-plane resolution of 0.39 × 0.39 mm. Image processing and analysis were performed on a SpecStation (SUN 4/110) using the M R I / I M A G E software (New Methods Research, St. Louis; release 1.5). Total brain planes and lesioned regions were quantified by using a segmentation function which automatically determines a threshold. Based on these thresholds automatic contour setting was performed, leading to the voxel sum in the respective regions of interest.
2.4. Histochemistry The extent of cerebral infarction was determined by a modification of the method of Bederson et al. (1986). After the rats were killed by an intracardiac injection (0.2 ml/animal) of T61 (Hoechst), the brains were rapidly removed and dissected coronally at 2 mm intervals, using a rodent brain slicer (Activational Systems Inc.; Michigan, USA). The brain slices were incubated in 1% TTC (2,3,5-triphenyltetrazolium chloride) in phosphate-buffered saline at 37°C for 45 min and stored in 4% buffered Formalin. Scaled photographs (Kodak Ektachrome 50) of the posterior surfaces of the fixed brain sections were quantified using a computerized image analysis system (Kontron CARDIO 200) to determine the area with deficient TTC staining as a percentage of the total area.
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2.5. Statistical analysis All results are presented as means +_ S.E.M. Comparison of means was performed among groups by using analysis of variance ( A N O V A ) add NewmanKeuls multiple range test. Student's paired t-test was used where appropriate. Differences were considered significant at P < 0.05.
2.6. Drugs T r e a t m e n t with levemopamil ((2S)-2-isopropyl-5(methyl-phenethylamino)-2-phenylvaleronitrile hydrochloride) consisted of three injections via .the tail vein given immediately after middle cerebral artery occlusion and 2 h and 4 h later (either 3 x 2 m g / k g or 3 × 4 m g / k g ) . The volume of each injection was 5 m l / k g and the duration was about 3 min. The controls were injected with equivalent volumes of saline solution (0.9% NaCI) at corresponding times.
Z Z Body temperature During surgery for middle cerebral artery occlusion, the rectal t e m p e r a t u r e of the anaesthetized animals was carefully controlled by a feedback regulated heating device. However, at later times of intravenous treatment with levemopamil (2 h and 4 h after middle cerebral artery occlusion), the animals were conscious. Hence, the highest dosage administered in our stroke experiments was tested for possible body temperature effects in six conscious rats. As a reference another group of animals (n = 6) was treated with the 5-HT~A receptor agonist 8 - O H - D P A T (8-hydroxy-2-(di-n-propylamino)-tetralin) in a manner (1 m g / k g s.c.) that is also capable of reducing infarct size in the rat (Bielenberg and Burckhardt, 1990).
3. Results 3.1. Effect of levemopamil on brain oedema Measurements of systolic blood pressure in several conscious S H R a few days before the experiments confirmed the hypertensive status of the rats; the m e a n value was 194 m m Hg (range 169-240 m m Hg). Focal cerebral o e d e m a was observed in the rat brains as sharply demarcated regions of high signal intensity (typically 80% signal increase compared to the contralateral side) in T2-weighted magnetic resonance images taken 24 h after p e r m a n e n t occlusion of the left middle cerebral artery (Figs. 1-4). O e d e m a always occurred in cortical and subcortical areas in the upper transversal brain plane ( + 6.5 m m dorsal to the interaural line, Figs. 1 and 3) and the adjacent lower brain
Fig. 1. Transversal T2-weighted proton spin echo image (TR/TE 2000/80 ms) of a rat brain (control, 0.9% NaC1 i.v.) obtained in vivo 24rh after middle cerebral artery occlusion. Stereotactic coordinates correspond to a plane + 6.5 mm dorsal to the interaural line. Note the region of oedema with high signal intensity and the midline shift due to the oedematous swelling of the left hemisphere (1: lateral ventricle, 2: caudate putamen, 3: frontal cortex, 4: parietal cortex, 5: temporal cortex, 6: occipital cortex, 7: rhinal cortex, 8: cerebellum).
plane ( + 4.5 m m dorsal to the interaural line, Figs. 2 a n d 4). As seen from the magnetic resonance images, focal o e d e m a was detected in the caudate putamen and the frontal, parietal, temporal and occipital cortices of the control animals. T r e a t m e n t with levemopamil, beginning immediately after middle cerebral artery occlusion and consisting of three slow intravenous injections each 2 h apart, led to significant dose-dependent diminutions of cerebral o e d e m a in both brain planes as summarized in Table 1. In group 2, which was treated with a low dose of levemopamil, the extent of o e d e m a decreased from 21.2 + 1.7% to 16.9 + 0.8% ( P < 0.05) in the u p p e r brain plane and from 15.8+ 1.2% to 12.5 _ 0.6% ( P < 0.05) in the lower brain plane. Thus, the relative extent of o e d e m a was reduced in group 2 by 20 + 3.7% in the u p p e r and by 21 + 3.8% in the lower brain plane as compared to the mean value of the controls. The higher dose in group 3 provoked a decrease in the
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Fig. 2. Magnetic resonance image of the same rat as in Fig. 1 but taken in the transversal brain plane + 4.5 m m dorsal to the interaural line (1: 3rd ventricle, 2: lateral ventricle, 3: aqueduct, 4: caudate putamen, 5: insular cortex, 6: parietal cortex, 7: temporal cortex, 8: cerebellum).
Table 1 Effect of intravenous treatment with levemopamil on extent of oedema in spontaneously hypertensive rats Group
Transversal Total brain Extent of brain plane section oedema (mm) d (pixel) (pixel)
(1) Control b +6.5 (n = 16) +4.5 (2) Levemopamil e +6.5 3×2 mg/kg +4.5 (n = 17) (3) Levemopamil c +6.5 3×4mg/kg +4.5 (n = 13)
Extent of oedema (% of total brain section)
5674--+56 6125+43 5738+48 6248+23
1 192+88 21.2+ 1.7 964+74 15.8+1.2 9 7 2 + 4 8 a 16.9+0.8 a 7 8 4 + 3 9 a 12.5_+0.6 a
5992_+o'~ 6107_+63
891_+44 a 14.9_+0.7 a ' 659_+35 a 10.8_+0.6 a
Means _+S.E.M. (n = n u m b e r of animals) Level of significance was P < 0.05 (Newman-Keuls test) compared to control, b 3 × 5 m l / k g physiological NaC1. c Levemopamil was administered 0 h (immediately after middle cerebral artery occlusion), 2 h and 4 h after middle cerebral artery occlusion, d The numbers indicate the distance to the interaural line referring to the coordinates of the stereotactic atlas of Paxinos and Watson (1982). a
Fig. 3. T2-weighted magnetic resonance image as in Fig. 1 but obtained from a rat treated with levemopamil (3 X4 m g / k g ) . The arrow indicates the region of the posterior cortex where the o e d e m a reduction is most pronounced (see Fig. 1 for comparison).
extent of oedema from 21.2 _+ 1.7% to 14.9 + 0.7% (P < 0.05) in the upper brain plane and from 15.8 + 1.2% to 10.8 + 0.6% ( P < 0.05) in the lower brain plane. Hence, with a higher dose, reductions in the relative extent of oedema of 30 + 3.5% and 31 + 4.0% were achieved in the upper and lower brain planes, respectively. The oedema reduction by levemopamil was most pronounced in the temporal and occipital cortices (Figs. 3 and 4).
3.2. Effect of levemopamil on brain infarction Reductions in infarct size were observed by TTCstaining in all brain planes of rats treated with the low dose (3 × 2 mg/kg) of levemopamil (Fig. 5). This cerebroprotective effect was amplified in animals by intravenous treatment with a higher dosage (3 × 4 mg/kg) of levemopamil. In both treatment groups the diminution of infarct size was most pronounced in the periphery of the territory supplied by the middle cerebral artery. Thus, dose-dependent infarct reductions of up to 39 + 12% and 48 + 11% were measured in the pos-
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Changein rectaltemperature(°C) 0.5-
-0.5 -1.0
-
*
-1.5-
* ~
_, /
-2.0-2.5 -3.0
0
3~0
6~0
9'0 1;20 Timeafter injection(min)
Fig. 6. Body temperature of conscious spontaneously hypertensive rats treated with levemopamil (4 m g / k g i.v., n = 6) or 8 - O H - D P A T (1 m g / k g s.c., n = 6). M e a n + S.E.M., * P < 0.05 versus body temperature before treatment (Student's paired t-test).
terior brain plane 3 of animals treated with the low and high doses of levemopamil, respectively.
3.3. Effect of levemopamil on body temperature Fig. 4. Ta-weighted magnetic resonance image in the adjacent lower brain plane of the same rat as in Fig. 3. The arrow denotes again the area of the cerebral cortex where the o e d e m a reduction is most obvious in levemopamil-treated rats as compared to the controls (Fig. 2).
% Controlinfarctarea
The results of the body temperature measurements in conscious spontaneously hypertensive rats are shown in Fig. 6. No significant changes in body temperature were observed in levemopamil-treated animals. In contrast, a marked and sustained decrease in body temperature was manifested in animals treated with the reference compound 8-OH-DPAT, as could be expected from the literature (Hjorth, 1985).
1°°1 8O
4. Discussion
60
40I 20
0
3
5 7 9 11 Brain plane (ram from interaural linie)
Fig. 5. Extent of infarction as determined by TI'C-staining in coronal brain planes of spontaneously hypertensive rats treated intravenously with 3 X 2 m g / k g (hatched bars) or 3 × 4 m g / k g (open bars) of levemopamil. Values are expressed as percentages of the relative infarct areas of the untreated control rats ( = 100%) and brain planes are referred to the stereotactic atlas of Paxinos and Watson (1982). * P < 0.05, levemopamil-treated vs. untreated animals (NewmanKeuls multiple range test).
Intravenous treatment of focal cerebral ischaemia with levemopamil significantly reduced the extent of cerebral oedema, as evaluated in SHR by T2-weighted magnetic resonance imaging 24 h after middle cerebral artery occlusion. Furthermore, the diminution of oedema size was found to be dose-dependent in the different brain regions under investigation. The protective effects were most pronounced in posterior cortical regions. These oedema reductions represent direct effects of levemopamil and not indirect cerebroprotection mediated by drug-induced hypothermia. The body temperature of the rats was maintained constant by a feedback-controlled heating pad when levemopamil was administered immediately after middle cerebral artery occlusion. Further the levemopamil injections 2 h and
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4 h after middle cerebral artery occlusion would not have caused hypothermia because, unlike the 5HTIAagonist 8-OH-DPAT which also has the capacity to reduce infarct size in the rat middle cerebral artery occlusion model (Bielenberg and Burkhardt, 1990), levemopamil does not reduce body temperature in conscious animals. Thus, the mechanism of cerebroprotection by levemopamil is not generalized cooling of the animals which has been demonstrated for other pharmacological agents (Hall et al., 1993; Corbett et al., 1990; Buchan and Pulsinelli, 1990) and which may cause complications in febrile stroke patients. The results of our study indicate that the cerebroprotection provided by levemopamil treatment is not only achieved in the normotensive rat as has been shown earlier (Nakayama et al., 1988; Block et al., 1990; Lin et al., 1990; Morikawa et al., 1991) but also in the spontaneously hypertensive rat. Hypertension is considered to be one of the major risk factors for the occurence of stroke (Dunbabin and Sandercock, 1990). The efficacy of Ca 2+ antagonists in the treatment of stroke has been questioned when associated with chronic hypertension (Ginsberg and Busto, 1989). Infarct size may be reduced by Ca 2+ antagonists via dilatation of collateral blood vessels in the vicinity of the affected territory, thereby enhancing the nutrient supply to cells in the border zone of the ischaemic area. The capacity of collateral blood vessels to dilate in response to Ca 2+ antagonists may however be impaired in chronic hypertension due to changed vessel morphology as a consequence of the persistent high blood pressure (Baumbach and Heistad, 1988). Nevertheless, our results and also investigations by others (Dirnagl et al., 1990; Sauter and Rudin, 1986) demonstrate that pharmacological salvage of ischaemic brain tissue is also possible after permanent middle cerebral artery occlusion in hypertensive rats. The diminution of brain oedema was most pronounced in the border zone of the territory supplied by the middle cerebral artery and especially the posterior cortex. In these regions, infarct size, as determined histologically, was also most effectively diminished by levemopamil. A possible mechanism of drug action could be improved perfusion in the border zone of the ischaemic area because levemopamil has been shown to dilate preferentially cerebral blood vessels (Raschack et al., 1989) and to enhance cerebral blood flow after intravenous administration in rats (Szabo, 1989). Similar conclusions on the amelioration of tissue perfusion in the penumbra of the ischaemic area have been drawn by Rudin et al. (1991) from magnetic resonance perfusion studies with middle cerebral artery-occluded rats treated with the dihydropyridine Ca 2+ channel blocker isradipine. In addition to a blockade of Ca 2+ channels, the strong 5-HT 2 receptor antagonism of levemopamil may have contributed to the improved
tissue perfusion in brain ischaemia which is associated with an enhanced release of serotonin (Phebus and Clemens, 1989). Besides its pronounced vasodilating properties, levemopamil has shown remarkable metabolic effects in studies on global brain ischaemia in rats and cats. Amelioration of glucose utilization and accelerated restoration of high-energy phosphates have been observed during recovery from transient brain ischaemia in rats (Bielenberg et al., 1987; Szabo and Hofmann, 1989). In addition, reduced changes in tissue lactate and NADH concentrations during ischaemia and an earlier return to baseline levels thereafter have been found in cats (Kovach et al., 1988; Ligeti et al., 1989). It remains to be clarified, however, whether dilatation of collateral blood vessels or direct cytoprotection are the prime mechanisms in the amelioration of energy metabolism and reduction of oedema when cerebral ischaemia is treated with levemopamil in chronic hypertension. The combined use of 1 H / a l p magnetic resonance spectroscopy, diffusion-weighted magnetic resonance imaging and contrast-enhanced echo planar imaging, techniques which have become available, may help to answer this question by simultaneous measurements of energy metabolism, development of oedema and tissue perfusion (Kucharczyk et al., 1989; Minematsu et al., 1992). The development of ischaemic oedema was attenuated in a clinically relevant stroke model by treatment with levemopamil. Similarly, treatment with levemopamil has been shown to reduce cerebral oedema in experimental brain trauma (Okiyama et al., 1992). Investigations of behaviour and motor function have suggested that the benefit of a reduction of brain oedema may consist of significantly improved neurological outcome in both focal ischaemia and trauma (Okiyama et al., 1992; Tominaga and Ohnishi, 1989). Accordingly, the diminution of the extent of cerebral oedema by levemopamil demonstrated by quantitative magnetic resonance imaging may encourage clinical studies of the drug in cerebrovascular disorders.
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B. Elger et al. / European Journal of Pharmacology 254 (1994) 65-71 and on postischemic energy metabolism in the rat brain, J. Cereb. Blood Flow Metab. 7, 480. Bielenberg, G.W. and M. Burkhardt, 1990, 5-HydroxytryptaminelA agonists. A new therapeutic principle for stroke treatment, Stroke 21 (Suppl. 4), IV-161. Block, F., R.M.A. Jaspers, C. Helm and K.-H. Sontag, 1990, Semopamil ameliorates ischemic brain damage in rats: histological and behavioural approaches, Life Sci. 47, 1511. Buchan, A. and W.A. Pulsinelli, 1990, Hypothermia but not the N-methyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia, J. Neurosci. 10, 311. Corbett, D., S. Evans, C. Thomas, D. Wang and R.A. Jonas, 1990, MK-801 reduced cerebral ischemic injury by inducing hypothermia, Brain Res. 514, 300. Dirnagl, U., J. Tanabe and W. Pulsinelli, 1990, Pre- and post-treatment with MK-801 but not pretreatment alone reduces neocortical damage after focal cerebral ischemia in the rat, Brain Res. 527, 62. Dunbabin, D.W. and P.A.G. Sandercock, 1990, Preventing stroke by the modification of risk factors, Stroke 21 (Suppl. 4), IV-36. Duverger, D. and E.T. MacKenzie, 1988, The quantification of cerebral infarction following focal ischemia in the rat: influence of strain, arterial pressure, blood glucose concentration and age, J. Cereb. Blood Flow Metab. 8, 449. Gerold, M. and H. Tschirky, 1968, Measurement of blood pressure in unanaesthetized rats and mice, Arzneim. Forsch. 18, 1285. Ginsberg, M.D. and R. Busto, 1989, Rodent models of cerebral ischemia, Stroke 20, 1627. Goldstein, L.B. and J.N. Davis, 1990, Restorative neurology. Drugs and recovery following stroke, Stroke 21, 1636. Hall, E.D., P.K. Andrus and K.E. Pazara, 1993, Protective efficacy of a hypothermic pharmacological agent in gerbil forebrain ischemia, Stroke 24, 711. Hayes, C.E., W.A. Edelstein, J.F. Schenck, O.M. Mueller and M. Eash, 1985, An efficient, highly homogeneous radiofrequency coil for whole-body NMR imaging at 1.5 T, J. Magn. Reson. 63, 622. Hjorth, S., 1985, Hypothermia in the rat induced by the potent serotoninergic agent 8-OH-DPAT, J. Neural Transm. 61, 131. Klatzo, I., M. Seida, H.G. Wagner and P. Ting, 1986, Features of ischemic brain edema, in: Pharmacology of Cerebral Ischemia, ed. J. Krieglstein (Elsevier Science Publishers, Amsterdam) p. 23. Kovach, A.G.B., L.T. Nguyen, L. Pek, L. Dezsi and Z.S. Lohinai, 1988, The effect of calcium entry blocker S-emopamil on cerebrocortical metabolism and blood flow changes evoked by graded hypotension, in: Oxygen Transport to Tissues, eds. G. Biro and K. Rakusan, Vol. 13, 461. Kucharczyk, J., W. Chew, N. Derugin, M. Mosely, C. Rollin, I. Berry and D. Norman, 1989, Nicardipine reduces ischemic brain injury. Magnetic resonance imaging/spectroscopy study in cats, Stroke 20, 268. Kumami, K., T. Yamamoto, A. ViUacara, B.B. Mrsulja and M. Spatz, 1990, Ischemic cerebral edema. 5-hydroxytryptarnine receptors and the physical state of synaptosomal membranes, Adv. Neurol. 52, 47. Ligeti, L., M.D. Osbakken, H. Subramanian, J.S. Leigh, L. Dezsi, K. Ikrenyi, A.G.B. Kovach and B. Chance, 1989, Effect of emopamil on metabolic changes of brain ischemia, in: Pharmacology of Cerebral Ischemia, ed. J. Krieglstein (Wiss. Verl.-Ges., Stuttgart) p. 117. Lin, B., W.D. Dietrich, R. Busto and M.D. Ginsberg, 1990, (S)Emopamil protects against global ischemic brain injury in rats, Stroke 21, 1734. Minematsu, K., L. Li, C.H. Sotak, M.A. Davis and M. Fisher, 1992, Reversible focal ischemic injury demonstrated by diffusionweighted magnetic resonance imaging in rats, Stroke 23, 1304.
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Morikawa, E., M.D. Ginsberg, W.D. Dietrich, R.C. Duncan and R. Busto, 1991, Postischemic (S)-emopamil therapy ameliorates focal ischemic brain injury in rats, Stroke 22, 355. Nakayama, H., M.D. Ginsberg and W.D. Dietrich, 1988, (S)Emopamil, a novel calcium channel blocker and serotonin $2 antagonist, markedly reduces infarct size following middle cerebral artery occlusion in the rat, Neurology 38, 1667. Naruse, S., Y. Aoki, R. Takei, Y. Horikawa and S. Ueda, 1991, Effects of atrial natriuretic peptide on ischemic brain edema in rats evaluated by proton magnetic resonance method, Stroke 22, 61. Okiyama, K., D.H. Smith, M.J. Thomas and T.K. Mclntosh, 1992, Evaluation of a novel calcium channel blocker, (S)-emopamil, on regional cerebral edema and neurobehavioral function after experimental brain injury, J. Neurosurg. 77, 607. Paxinos, G. and C. Watson, 1982, The Rat Brain in Stereotactic Coordinates (Academic Press, Sydney, Australia). Petito, C.K., 1986, Experimental models of cerebral ischemia, in: Pharmacology of Cerebral Ischemia, ed. J. Krieglstein (Elsevier Science Publishers, Amsterdam) p. 111. Phebus, L.A. and J.A. Clemens, 1989, Effects of transient, global, cerebral ischemia on striatal extracellular dopamine, serotonin and their metabolities, Life Sci. 44, 1335. Raschack, M., L. Unger and L. Szabo, 1989, (S)-Emopamil, a brainpenetrating calcium antagonist of the verapamil group for the treatment of cerebrovascular disorders, in: Pharmacology of Cerebral Ischemia, ed. J. Krieglstein (Wiss. Verl.-Ges., Stuttgart) p. 103. Roussel, S., E. Pinard and J. Seylaz, 1992, Effect of MK-801 on focal brain infarction in normotensive and hypertensive rats, Hypertension 19, 40. Rudin, M., W. Zierhut, A. Sauter and N.S. Cook, 1991, New developments in cardiovascular magnetic resonance imaging and spectroscopy, Trends Pharmacol. Sci. 12, 416. Sauer, D., P.R. Allegrini, A. Cosenti, A. Pataki, H. Amacker and G.E. Fagg, 1993, Characterization of the cerebroprotective efficacy of the competitive NMDA receptor antagonist CGP 40116 in a rat model of focal cerebral ischemia: an in vivo magnetic resonance imaging study, J. Cereb. Blood Flow Metab. 13, 595. Sauter, A. and M. Rudin, 1986, Calcium antagonists reduce the extent of infarction in rat middle cerebral artery occlusion model as determined by quantitative magnetic resonance imaging, Stroke 17, 1228. Shaw, C.M., E.C. Alvord and R.G. Berry, 1959, Swelling of the brain following ischemic infarction with arterial occlusion, Arch. Neurol. 1, 161. SiesjS, B.K., 1986, Cell damage in the brain caused by ischemia: an overview, in: Pharmacology of Cerebral Ischemia, ed. J. Krieglstein (Elsevier Science Publishers) p. 3. Szabo, L., 1989, (S)-Emopamii, a novel calcium and serotonin antagonist for the treatment of cerebrovascular disorders. 2nd communication: Brain penetration, cerebral vascular and metabolic effects, Arzneim. Forsch./Drug Res. 39, 309. Szabo, L. and H.P. Hofmann, 1989, (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, Arzneim. Forsch./Drug Res. 39, 314. Tamura, A., D.I. Graham, J. McCulloch and G.M. Teasdale, 1981, 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. Tominaga, T. and S.T. Ohnishi, 1989, Interrelationship of brain edema, motor deficits, and memory impairment in rats exposed to focal ischemia, Stroke 20, 513.
European Journal of Pharmacology254 (1994) 65-71
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Oedema reduction by levemopamil in focal cerebral ischaemia of spontaneously hypertensive rats studied by magnetic resonance imaging Bernd Elger *, Jiirgen Seega, Manfred Raschack Research and Development, Knoll AG, P.O. Box 21 08 05, D-67008 Ludwigshafen, Germany
(Received 12 July 1993;revised MS received 6 December 1993; accepted 10 December 1993)
Abstract The effect of treatment with the C a 2+ channel blocker and 5-HT2 receptor antagonist levemopamil (recommended INN for (S)-emopamil) on the extent of ischaemic brain oedema was studied by magnetic resonance imaging in vivo. Focal cerebral ischaemia was induced in spontaneously hypertensive rats by permanent middle cerebral artery occlusion. The treatment consisted of slow intravenous injections of an aqueous solution of levemopamil given immediately after middle cerebral artery occlusion and again 2 h and 4 h later. One group of animals (n = 17) received 3 × 2 mg/kg of levemopamil (total dose: 6 mg/kg) and another group (n = 13) received 3 × 4 mg/kg (total dose: 12 mg/kg). Saline was administered to the controls (n = 16) at corresponding times. High-resolution T2-weighted spin echo images were obtained 24 h after middle cerebral artery occlusion from two transversal brain planes (4.5 mm and 6.5 mm dorsal to the interaural line). Dose-dependent reductions of brain oedema were achieved in both brain planes. The lower dose of levemopamil reduced the extent of oedema significantly (P < 0.05) by 20 + 3.7% in the upper and by 21 + 3.8% in the lower brain plane as compared to the controls (means + S.E.M.). The higher dose diminished the extent of oedema in the same planes by 30 + 3.5% and 31 + 4.0%, respectively. Dose-dependent reductions of infarct size, as determined by vital tissue staining using 2,3,5-triphenyltetrazolium chloride (TTC), were observed in the levemopamil-treated groups. Body temperature was not affected by levemopamil, suggesting direct cerebroprotection by this drug. The results of this magnetic resonance imaging study indicate that the extent of brain oedema and infarction is dose dependently reduced by treatment with levemopamil in a clinically relevant stroke model. Key words: Magnetic resonance imaging; NMR tomography; Brain edema; Stroke; 5-HT receptor antagonist; Ca 2+ channel
blocker
I. Introduction Clinically, the formation of oedema in cerebral disorders such as stroke and trauma is a major complicating factor which causes aggravation of the diseases (Goldstein and Davis, 1990). An increased tissue pressure in oedematous areas may reduce blood flow in the adjacent tissues and thus extend the ischaemically affected territory (Klatzo et al., 1986). Furthermore, oedema may influence the neuronal control of vegetative function by compression in areas remote from the lesion and thereby cause the death of patients (Shaw et al., 1959). Evidence is accumulating that serotonin is
* Corresponding author. Tel. (0621) 589-2802, fax (0621) 589-2050. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0014-2999(93)E0887-X
involved in the formation of ischaemic cerebral oedema, as has recently been reviewed by Kumami et al. (1990). During brain ischaemia there is a marked increase in extracellular serotonin levels (Phebus and Clemens, 1989). Thus, the aim of the present study was to investigate the effects of levemopamil (recommended INN for (S)-emopamil) on the extent of oedema in focal cerebral ischaemia. This agent is a stereoselective antagonist of the cerebral 5-HT 2 receptor, having a g i of 4.4 n m o l / l for ketanserin displacement (Raschack et al., 1989). Other features are L-type Ca2+-channel antagonism, high cerebral availability and lower cardiovascular activity than e.g. the Ca 2÷ channel blocker verapamil. Middle cerebral artery occlusion in rats was used as a widely accepted clinically relevant animal model for human stroke (Petito, 1986; Siesj6, 1986;
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Ginsberg and Busto, 1989). However, in contrast to earlier studies on the beneficial effects of levemopamil in this model (Nakayama et al., 1988; Morikawa et al., 1991), we performed the experiments with spontaneously hypertensive rats (SHR) instead of normotensive rats for two reasons. Firstly, infarct variability is much lower in SHR than in normotensive rats (Duverger and MacKenzie, 1988). Secondly, stroke frequently occurs in hypertensive patients (Dunbabin and Sandercock, 1990). Since significant infarct reduction may be found only in normotensive rats but not in SHR, as has been shown for the N-methyl-D-aspartate antagonist MK-801 (Roussel et al., 1992), positive results obtained with SHR may aid in the initiation of clinical drug testing. The influence of intravenous treatment with levemopamil on brain oedema was measured by magnetic resonance imaging in vivo. Magnetic resonance imaging is a widely used technique for medical diagnosis and, furthermore, it is well suited to determine drug effects on ischaemic brain oedema (Kucharczyk et al., 1989; Naruse et al., 1991; Allegrini and Sauer, 1992; Sauer et al., 1993). T2-weighted spin echo imaging was applied to display the spatial extent of brain oedema because a high correlation between tissue water content and oedematous area on T 2weighted images has been demonstrated (Naruse et al., 1991). With regard to clinical stroke therapy, the intravenous route was chosen in the present study and different doses of levemopamil were tested.
2. Materials and methods
Our experiments were performed in accordance to the requirements of the German laws for the protection of animals (Deutsches Tierschutzgesetz).
2.1. Animals Male SHR (280-300 g) were obtained from Mollegaards (Skensved, Denmark) and maintained under standard conditions (12 h day/night cycle, 22°C, 50% humidity) with free access to food and tap water before and after surgery. Systolic arterial blood pressure of conscious animals was measured by tail-cuff plethysmography using a piezo crystal a few days before surgery as described elsewhere (Gerold and Tschirky, 1968). Rectal temperature was determined with a thermoelectrical probe.
2.2. Surgery The animals were anaesthetized with sodium pentobarbital (60 mg/kg i.p.) and placed on a thermostatically regulated heating pad to maintain rectal temperature at 37°C. The permanent occlusion of the left
proximal middle cerebral artery was performed by the method of Tamura et al. (1981).
2.3. Magnetic resonance imaging The animals were anaesthetized with sodium pentobarbital (60 mg/kg i.p.) for magnetic resonance imaging 24 h after middle cerebral artery occlusion. Experiments were carried out in vivo with a General Electric CSI-II 2.0 Tesla NMR system equipped with Acustar self-shielded gradient coils (maximum gradient strength + 20 gauss/cm, 15 cm clear bore). Magnetic resonance imaging was performed by 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., 1985). The animals were placed in a home-built plexiglas stereotactic holder fitting into the birdcage resonator. 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. T2-weighted images (TR = 2 s, TE = 80 ms, NA = 4) were taken of transversal brain planes located +4.5 mm and + 6.5 mm dorsal to the interaural line. The stereotactic coordinates are from the atlas of Paxinos and Watson (1982). Further measurements dorsal and ventral to these two adjacent planes were not performed to avoid erroneous overestimation of oedema due to partial volume effects. Images displayed 2-mm thick slices with an in-plane resolution of 0.39 × 0.39 mm. Image processing and analysis were performed on a SpecStation (SUN 4/110) using the M R I / I M A G E software (New Methods Research, St. Louis; release 1.5). Total brain planes and lesioned regions were quantified by using a segmentation function which automatically determines a threshold. Based on these thresholds automatic contour setting was performed, leading to the voxel sum in the respective regions of interest.
2.4. Histochemistry The extent of cerebral infarction was determined by a modification of the method of Bederson et al. (1986). After the rats were killed by an intracardiac injection (0.2 ml/animal) of T61 (Hoechst), the brains were rapidly removed and dissected coronally at 2 mm intervals, using a rodent brain slicer (Activational Systems Inc.; Michigan, USA). The brain slices were incubated in 1% TTC (2,3,5-triphenyltetrazolium chloride) in phosphate-buffered saline at 37°C for 45 min and stored in 4% buffered Formalin. Scaled photographs (Kodak Ektachrome 50) of the posterior surfaces of the fixed brain sections were quantified using a computerized image analysis system (Kontron CARDIO 200) to determine the area with deficient TTC staining as a percentage of the total area.
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2.5. Statistical analysis All results are presented as means +_ S.E.M. Comparison of means was performed among groups by using analysis of variance ( A N O V A ) add NewmanKeuls multiple range test. Student's paired t-test was used where appropriate. Differences were considered significant at P < 0.05.
2.6. Drugs T r e a t m e n t with levemopamil ((2S)-2-isopropyl-5(methyl-phenethylamino)-2-phenylvaleronitrile hydrochloride) consisted of three injections via .the tail vein given immediately after middle cerebral artery occlusion and 2 h and 4 h later (either 3 x 2 m g / k g or 3 × 4 m g / k g ) . The volume of each injection was 5 m l / k g and the duration was about 3 min. The controls were injected with equivalent volumes of saline solution (0.9% NaCI) at corresponding times.
Z Z Body temperature During surgery for middle cerebral artery occlusion, the rectal t e m p e r a t u r e of the anaesthetized animals was carefully controlled by a feedback regulated heating device. However, at later times of intravenous treatment with levemopamil (2 h and 4 h after middle cerebral artery occlusion), the animals were conscious. Hence, the highest dosage administered in our stroke experiments was tested for possible body temperature effects in six conscious rats. As a reference another group of animals (n = 6) was treated with the 5-HT~A receptor agonist 8 - O H - D P A T (8-hydroxy-2-(di-n-propylamino)-tetralin) in a manner (1 m g / k g s.c.) that is also capable of reducing infarct size in the rat (Bielenberg and Burckhardt, 1990).
3. Results 3.1. Effect of levemopamil on brain oedema Measurements of systolic blood pressure in several conscious S H R a few days before the experiments confirmed the hypertensive status of the rats; the m e a n value was 194 m m Hg (range 169-240 m m Hg). Focal cerebral o e d e m a was observed in the rat brains as sharply demarcated regions of high signal intensity (typically 80% signal increase compared to the contralateral side) in T2-weighted magnetic resonance images taken 24 h after p e r m a n e n t occlusion of the left middle cerebral artery (Figs. 1-4). O e d e m a always occurred in cortical and subcortical areas in the upper transversal brain plane ( + 6.5 m m dorsal to the interaural line, Figs. 1 and 3) and the adjacent lower brain
Fig. 1. Transversal T2-weighted proton spin echo image (TR/TE 2000/80 ms) of a rat brain (control, 0.9% NaC1 i.v.) obtained in vivo 24rh after middle cerebral artery occlusion. Stereotactic coordinates correspond to a plane + 6.5 mm dorsal to the interaural line. Note the region of oedema with high signal intensity and the midline shift due to the oedematous swelling of the left hemisphere (1: lateral ventricle, 2: caudate putamen, 3: frontal cortex, 4: parietal cortex, 5: temporal cortex, 6: occipital cortex, 7: rhinal cortex, 8: cerebellum).
plane ( + 4.5 m m dorsal to the interaural line, Figs. 2 a n d 4). As seen from the magnetic resonance images, focal o e d e m a was detected in the caudate putamen and the frontal, parietal, temporal and occipital cortices of the control animals. T r e a t m e n t with levemopamil, beginning immediately after middle cerebral artery occlusion and consisting of three slow intravenous injections each 2 h apart, led to significant dose-dependent diminutions of cerebral o e d e m a in both brain planes as summarized in Table 1. In group 2, which was treated with a low dose of levemopamil, the extent of o e d e m a decreased from 21.2 + 1.7% to 16.9 + 0.8% ( P < 0.05) in the u p p e r brain plane and from 15.8+ 1.2% to 12.5 _ 0.6% ( P < 0.05) in the lower brain plane. Thus, the relative extent of o e d e m a was reduced in group 2 by 20 + 3.7% in the u p p e r and by 21 + 3.8% in the lower brain plane as compared to the mean value of the controls. The higher dose in group 3 provoked a decrease in the
B. EIger et al. / European Journal of Pharmacology 254 (1994) 65-71
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Fig. 2. Magnetic resonance image of the same rat as in Fig. 1 but taken in the transversal brain plane + 4.5 m m dorsal to the interaural line (1: 3rd ventricle, 2: lateral ventricle, 3: aqueduct, 4: caudate putamen, 5: insular cortex, 6: parietal cortex, 7: temporal cortex, 8: cerebellum).
Table 1 Effect of intravenous treatment with levemopamil on extent of oedema in spontaneously hypertensive rats Group
Transversal Total brain Extent of brain plane section oedema (mm) d (pixel) (pixel)
(1) Control b +6.5 (n = 16) +4.5 (2) Levemopamil e +6.5 3×2 mg/kg +4.5 (n = 17) (3) Levemopamil c +6.5 3×4mg/kg +4.5 (n = 13)
Extent of oedema (% of total brain section)
5674--+56 6125+43 5738+48 6248+23
1 192+88 21.2+ 1.7 964+74 15.8+1.2 9 7 2 + 4 8 a 16.9+0.8 a 7 8 4 + 3 9 a 12.5_+0.6 a
5992_+o'~ 6107_+63
891_+44 a 14.9_+0.7 a ' 659_+35 a 10.8_+0.6 a
Means _+S.E.M. (n = n u m b e r of animals) Level of significance was P < 0.05 (Newman-Keuls test) compared to control, b 3 × 5 m l / k g physiological NaC1. c Levemopamil was administered 0 h (immediately after middle cerebral artery occlusion), 2 h and 4 h after middle cerebral artery occlusion, d The numbers indicate the distance to the interaural line referring to the coordinates of the stereotactic atlas of Paxinos and Watson (1982). a
Fig. 3. T2-weighted magnetic resonance image as in Fig. 1 but obtained from a rat treated with levemopamil (3 X4 m g / k g ) . The arrow indicates the region of the posterior cortex where the o e d e m a reduction is most pronounced (see Fig. 1 for comparison).
extent of oedema from 21.2 _+ 1.7% to 14.9 + 0.7% (P < 0.05) in the upper brain plane and from 15.8 + 1.2% to 10.8 + 0.6% ( P < 0.05) in the lower brain plane. Hence, with a higher dose, reductions in the relative extent of oedema of 30 + 3.5% and 31 + 4.0% were achieved in the upper and lower brain planes, respectively. The oedema reduction by levemopamil was most pronounced in the temporal and occipital cortices (Figs. 3 and 4).
3.2. Effect of levemopamil on brain infarction Reductions in infarct size were observed by TTCstaining in all brain planes of rats treated with the low dose (3 × 2 mg/kg) of levemopamil (Fig. 5). This cerebroprotective effect was amplified in animals by intravenous treatment with a higher dosage (3 × 4 mg/kg) of levemopamil. In both treatment groups the diminution of infarct size was most pronounced in the periphery of the territory supplied by the middle cerebral artery. Thus, dose-dependent infarct reductions of up to 39 + 12% and 48 + 11% were measured in the pos-
B. Elger et aL / European Journal of Pharmacology 254 (1994) 65-71
69
Changein rectaltemperature(°C) 0.5-
-0.5 -1.0
-
*
-1.5-
* ~
_, /
-2.0-2.5 -3.0
0
3~0
6~0
9'0 1;20 Timeafter injection(min)
Fig. 6. Body temperature of conscious spontaneously hypertensive rats treated with levemopamil (4 m g / k g i.v., n = 6) or 8 - O H - D P A T (1 m g / k g s.c., n = 6). M e a n + S.E.M., * P < 0.05 versus body temperature before treatment (Student's paired t-test).
terior brain plane 3 of animals treated with the low and high doses of levemopamil, respectively.
3.3. Effect of levemopamil on body temperature Fig. 4. Ta-weighted magnetic resonance image in the adjacent lower brain plane of the same rat as in Fig. 3. The arrow denotes again the area of the cerebral cortex where the o e d e m a reduction is most obvious in levemopamil-treated rats as compared to the controls (Fig. 2).
% Controlinfarctarea
The results of the body temperature measurements in conscious spontaneously hypertensive rats are shown in Fig. 6. No significant changes in body temperature were observed in levemopamil-treated animals. In contrast, a marked and sustained decrease in body temperature was manifested in animals treated with the reference compound 8-OH-DPAT, as could be expected from the literature (Hjorth, 1985).
1°°1 8O
4. Discussion
60
40I 20
0
3
5 7 9 11 Brain plane (ram from interaural linie)
Fig. 5. Extent of infarction as determined by TI'C-staining in coronal brain planes of spontaneously hypertensive rats treated intravenously with 3 X 2 m g / k g (hatched bars) or 3 × 4 m g / k g (open bars) of levemopamil. Values are expressed as percentages of the relative infarct areas of the untreated control rats ( = 100%) and brain planes are referred to the stereotactic atlas of Paxinos and Watson (1982). * P < 0.05, levemopamil-treated vs. untreated animals (NewmanKeuls multiple range test).
Intravenous treatment of focal cerebral ischaemia with levemopamil significantly reduced the extent of cerebral oedema, as evaluated in SHR by T2-weighted magnetic resonance imaging 24 h after middle cerebral artery occlusion. Furthermore, the diminution of oedema size was found to be dose-dependent in the different brain regions under investigation. The protective effects were most pronounced in posterior cortical regions. These oedema reductions represent direct effects of levemopamil and not indirect cerebroprotection mediated by drug-induced hypothermia. The body temperature of the rats was maintained constant by a feedback-controlled heating pad when levemopamil was administered immediately after middle cerebral artery occlusion. Further the levemopamil injections 2 h and
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4 h after middle cerebral artery occlusion would not have caused hypothermia because, unlike the 5HTIAagonist 8-OH-DPAT which also has the capacity to reduce infarct size in the rat middle cerebral artery occlusion model (Bielenberg and Burkhardt, 1990), levemopamil does not reduce body temperature in conscious animals. Thus, the mechanism of cerebroprotection by levemopamil is not generalized cooling of the animals which has been demonstrated for other pharmacological agents (Hall et al., 1993; Corbett et al., 1990; Buchan and Pulsinelli, 1990) and which may cause complications in febrile stroke patients. The results of our study indicate that the cerebroprotection provided by levemopamil treatment is not only achieved in the normotensive rat as has been shown earlier (Nakayama et al., 1988; Block et al., 1990; Lin et al., 1990; Morikawa et al., 1991) but also in the spontaneously hypertensive rat. Hypertension is considered to be one of the major risk factors for the occurence of stroke (Dunbabin and Sandercock, 1990). The efficacy of Ca 2+ antagonists in the treatment of stroke has been questioned when associated with chronic hypertension (Ginsberg and Busto, 1989). Infarct size may be reduced by Ca 2+ antagonists via dilatation of collateral blood vessels in the vicinity of the affected territory, thereby enhancing the nutrient supply to cells in the border zone of the ischaemic area. The capacity of collateral blood vessels to dilate in response to Ca 2+ antagonists may however be impaired in chronic hypertension due to changed vessel morphology as a consequence of the persistent high blood pressure (Baumbach and Heistad, 1988). Nevertheless, our results and also investigations by others (Dirnagl et al., 1990; Sauter and Rudin, 1986) demonstrate that pharmacological salvage of ischaemic brain tissue is also possible after permanent middle cerebral artery occlusion in hypertensive rats. The diminution of brain oedema was most pronounced in the border zone of the territory supplied by the middle cerebral artery and especially the posterior cortex. In these regions, infarct size, as determined histologically, was also most effectively diminished by levemopamil. A possible mechanism of drug action could be improved perfusion in the border zone of the ischaemic area because levemopamil has been shown to dilate preferentially cerebral blood vessels (Raschack et al., 1989) and to enhance cerebral blood flow after intravenous administration in rats (Szabo, 1989). Similar conclusions on the amelioration of tissue perfusion in the penumbra of the ischaemic area have been drawn by Rudin et al. (1991) from magnetic resonance perfusion studies with middle cerebral artery-occluded rats treated with the dihydropyridine Ca 2+ channel blocker isradipine. In addition to a blockade of Ca 2+ channels, the strong 5-HT 2 receptor antagonism of levemopamil may have contributed to the improved
tissue perfusion in brain ischaemia which is associated with an enhanced release of serotonin (Phebus and Clemens, 1989). Besides its pronounced vasodilating properties, levemopamil has shown remarkable metabolic effects in studies on global brain ischaemia in rats and cats. Amelioration of glucose utilization and accelerated restoration of high-energy phosphates have been observed during recovery from transient brain ischaemia in rats (Bielenberg et al., 1987; Szabo and Hofmann, 1989). In addition, reduced changes in tissue lactate and NADH concentrations during ischaemia and an earlier return to baseline levels thereafter have been found in cats (Kovach et al., 1988; Ligeti et al., 1989). It remains to be clarified, however, whether dilatation of collateral blood vessels or direct cytoprotection are the prime mechanisms in the amelioration of energy metabolism and reduction of oedema when cerebral ischaemia is treated with levemopamil in chronic hypertension. The combined use of 1 H / a l p magnetic resonance spectroscopy, diffusion-weighted magnetic resonance imaging and contrast-enhanced echo planar imaging, techniques which have become available, may help to answer this question by simultaneous measurements of energy metabolism, development of oedema and tissue perfusion (Kucharczyk et al., 1989; Minematsu et al., 1992). The development of ischaemic oedema was attenuated in a clinically relevant stroke model by treatment with levemopamil. Similarly, treatment with levemopamil has been shown to reduce cerebral oedema in experimental brain trauma (Okiyama et al., 1992). Investigations of behaviour and motor function have suggested that the benefit of a reduction of brain oedema may consist of significantly improved neurological outcome in both focal ischaemia and trauma (Okiyama et al., 1992; Tominaga and Ohnishi, 1989). Accordingly, the diminution of the extent of cerebral oedema by levemopamil demonstrated by quantitative magnetic resonance imaging may encourage clinical studies of the drug in cerebrovascular disorders.
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