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Dextromethorphan alters cerebral blood flow and protects against cerebral injury following focal ischemia
Neuroscience Letters, 133 (1991) 225-228
225
© 1991 ElsevierScientific Publishers Ireland Ltd. All rights reserved0304-3940/91/$ 03.50 NSL08233
Dextromethorphan alters cerebral blood flow and protects against cerebral injury following focal ischemia G a r y K . Steinberg, E n g H . L o , D a v i d M. K u n i s , G e r a l d A. G r a n t , Alex P o l j a k a n d R o b e r t D e L a P a z Department of Neurosurgery and Division of Neuroradiology, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.)
(Received 28 May 1991; Revisedversion received 20 August 1991; Accepted 28 August 1991) Key words: Dextromethorphan; Cerebral blood flow; Cerebral ischemia; N-Methyl-D-aspartate;Glutamate
The effectsof the N-methyl-o-aspartate (NMDA) antagonist dextromethorphan (DM) on regional cerebral blood flow (rCBF) and cerebral injury were studied in a rabbit model of transient focal ischemia. Anesthetized rabbits underwent 2 h occlusion of the left internal carotid, middle cerebral and anterior cerebral artery, followedby 4 h of reperfusion. Ten minutes after the onset of ischemia they were treated with either i.v. DM 20 mg/kg followed by 10 mg/kg/h (n =6) or normal saline (NS, n = 5). Control rabbits received DM (n = 3) or NS (n = 2) infusion without arterial occlusion. DM attenuated the sharp, post-ischemicrise in rCBF seen during reperfusion within the ischemiccore of NS controls (DM 31 ~; pre-ischemicvalue, NS 92%). DM also improved the delayed post-ischemic hypoperfusion compared with controls. DM infusion without arterial occlusion did not change rCBF values. Compared with NS controls, DM treated animals demonstrated recovery of the somatosensory evoked potential (DM 96% pre-ischemic values, NS 24%), 76% reduction in cortical edema and 92% decrease in cortical ischemic neuronal damage. We conclude that DM's effect on CBF may contribute to its neuroprotective action.
Dextromethorphan (D- 3-methoxy-N-methylmorphinan; D M ) is an opioid compound, but devoid of most narcotic properties [8]. D M is a noncompetitive, Nmethyl-D-aspartate ( N M D A ) antagonist [9, 12] that attenuates glutamate toxicity, N M D A - i n d u c e d damage and hypoxic injury in neuronal culture [3, 6]. D M also prevents ischemic neuronal damage and edema in animal models of focal cerebral ischemia when administered before ischemia or in a delayed manner after ischemia [1619]. It has been postulated that the neuroprotective effects of D M and other N M D A antagonists are related to blockade of neuronal N M D A receptors [17, 18]. However, it is possible that D M and these other N M D A antagonists provide neuroprotection by altering cerebral blood flow or metabolism. We undertook this study to investigate the effects of D M on regional cerebral blood flow (rCBF) and cerebral injury in a rabbit model of transient focal i schemia [17-19]. Male New Zealand white rabbits (2.5-3.5 kg) were anesthetized with 3 % halothane delivered by mask. After catheterization of the femoral artery and ear vein, the rabbits were tracheostomized, immobilized with 0.2 mg/ kg i.v. pancuronium bromide and artificially ventilated. Correspondence: G.K. Steinberg, Department of Neurosurgery S006, Stanford University Medical Center, Stanford, CA 94305, U.S.A.
Anesthesia was maintained with 0.5% halothane in 0.5 l/min 02:4.5 l/min air. Mean arterial pressure (MAP), end-expired CO2, heart rate, rectal temperature and temporalis muscle temperature were measured continuously. Arterial blood gasses, hematocrit and blood glucose were monitored intermittently. Rectal temperature was controlled at 39°C and temporalis muscle temperature at 37.5°C using a heating lamp and heating pad. Endexpired CO2 was maintained at 30-35 m m Hg by adjusting the ventilation rate. Arterial base deficit was corrected as necessary with i.v. sodium bicarbonate. After immobilization of the rabbit head in a stereotactic frame, the dorsal scalp was removed and somatosensory evoked potential (SEP) bone-screw electrodes were inserted 5 m m lateral (left and right) to bregma. Stimulating electrodes were placed over left and right median nerves. Square-wave stimuli (10 mA, 0.25 ms) were delivered at 4.1 Hz and SEPs were recorded with a Nicolet Pathfinder II electrodiagnostic system. One hundred and fifty responses were averaged for each measurement. SEP amplitudes were measured from the peak of the first major positive deflection to the trough of the adjacent negative deflection [18]. F o r analysis, SEP amplitudes were expressed as percentages of pre-ischemic values. To measure rCBF, two windows (2 m m diameter) were drilled over the left hemisphere: the first 15 m m anterior and 15 m m lateral to bregma, the second 0 m m anterior
226
and l0 mm lateral to bregma. The dura was left intact to prevent injury to the brain. A saline drip was also used during drilling to prevent overheating of the underlying brain. Laser-Doppler flow probes (TSI BPM 403A) were placed over the rCBF windows and using a micromanipulator were advanced under microscopic guidance to the surface of the brain. Care was taken not to damage the brain while adjusting the probe position. Since the laser-Doppler method has been demonstrated to provide accurate measurements of rCBF changes, but not absolute flow [5], all measurements of rCBF in this study were expressed as relative percentages of pre-ischemic or pre-drug values. In order to insure a uniform degree of pretreatment ischemia in all rabbits, we only used animals in which the SEPs were completely abolished within 10 min of arterial occlusion. Eleven rabbits underwent transorbital clip occlusion of the left internal carotid, proximal left anterior cerebral and proximal left middle cerebral arteries for 2 h, followed by 4 h of reperfusion. During the 2 h occlusion period, the MAP was kept 60-70 mm Hg by adjusting the halothane concentration between 0.5-0.7%. A loading dose of DM (20 mg/kg) followed by maintenance doses of 10 mg/kg/h (n=6) or an equivalent volume of normal saline (NS; n = 5) was blindly administered starting 10 min after occlusion. In order to examine DM's effects on rCBF in the non-ischemic brain, 5 anesthetized rabbits were administered i.v. DM (n = 3) or NS (n=2) without undergoing arterial occlusion. The dose of DM and experimental techniques were otherwise identical to those for the ischemic rabbits. Rabbits were sacrificed with sodium pentobarbital and perfused with saline followed by 10% formalin [17-19]. The brains were kept in the skull overnight, then removed and stored in formalin. Magnetic resonance imaging (MRI) studies were performed using sequences described previously (i .5 Tesla General Electric Signal Systems, 3 mm slice thickness, TR 2500, TE 100) [19]. Using 5 standard coronal images, abnormal high intensity areas were expressed as a percentage of total cortical or hemisphere area. Previous studies have shown that regions of abnormal MRI signal represent areas ofcytotoxic and vasogenic edema [4]. Histological examination of 5 standard coronal, hematoxylin-eosin stained sections (paraffin embedded, 6 #m thick) were performed to measure areas of ischemic neuronal damage (IND) [19]. An average percentage area ischemic edema or IND for each animal was calculated as the total area of edema or IND for all 5 coronal levels divided by the total area of cortex or hemisphere for all 5 levels. Statistical analyses utilized non-parametric Mann-Whitney U-tests and Wilcoxon sign tests; P < 0.05 was considered significant. There were no differences between groups in any syste-
mic parameters before arterial occlusion, during arterial occlusion or during reperfusion. These included rectal and temporalis muscle temperature, MAP, heart rate, paO2, paC02, pH, hematocrit, glucose, halothane administered, or volume of i.v. fluid infused. Laser-Doppler measurements demonstrated an immediate drop in rCBF after occlusion, however, not all rCBF windows showed similar degrees of reduced rCBF, demonstrating the variable distribution of the ischemic lesion. For this analysis, only sites where rCBF decreased below 20 % of pre-ischemic values (ischemic core) were used. In the DM group, one rabbit showed a de-
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Fig. I. Regional cerebral blood flow (rCBF) in dextromethorphan (DM) or normal saline (NS) treated rabbits. A: after transient focal ischemia, dextromethorphan prevents the post-ischemic rise in rCBF with the onset of reperfusion. D M also improves the delayed postisehemic hypoperfusion observed in the NS group. N u m b e r of rCBF window measurements at each time point (DM, n = 7; NS, n = 7). B: in non-ischemic rabbits rCBF does not differ between D M and NS groups (DM, n = 6 rCBF windows; NS, n = 4 rCBF windows). Mean _+ S.E.M. * P < 0 . 0 5 M a n n Whitney U-test for comparison between DM and NS. t P < 0 . 0 5 Wilcoxon signed rank test for comparison with pre-occlusion rCBF values.
227
crease in rCBF below 20~ in both windows and 5 rabbits showed decreases in only one window. In the NS group, two rabbits showed decreases below 20 % in both windows and 3 rabbits showed decreases below 20% in only one window. During the 2 h occlusion period, rCBF remained severely depressed, and no significant differences were found between the DM and NS groups (Fig. 1A). However, significant differences in rCBF patterns were observed during the reperfusion period. The NS group demonstrated an immediate, sharp post-ischemic rise in rCBF (92% of pre-ischemic values at 0.5 h of reperfusion), followed by a gradual deterioration to 35 % pre-ischemic values at 4 h of reperfusion. In the DM group, the immediate rise in rCBF after unocclusion was eliminated (P<0.05 at 0.5 h of reperfusion compared with NS controls), and rCBF gradually recovered to preischemic values at 4 h of reperfusion (Fig. 1A). In the NS group, rCBF values during the later stages of reperfusion (5 and 5.5 h) were significantly lower than preocclusion values ( P < 0.05, Wilcoxon signed rank test). rCBF in the DM group at these time points was not significantly different from pre-occlusion values. In the non-ischemic rabbits, rCBF declined slightly over the 6 h experiment, but did not differ between DM and NS groups (Fig. 1B). SEP amplitudes were reduced to zero between 5 and 10 min after onset of ischemia in all rabbits within both DM and NS groups. After reperfusion, SEP amplitudes in the DM group showed greater recovery than the NS group (Fig. 2). In the DM group, SEP recovered to 96.3 ___ 21.3% (mean -t- S.E.M.) of pre-ischemic values, whereas in the NS groups, SEP amplitudes were 23.8 + 16.9 % of pre-ischemic values (P < 0.05). In non-ischemic rabbits, SEP amplitudes did not change significantly with either DM or NS treatment. MRI analysis demonstrated 76% reduction in cortical ischemic edema and 79 % attenuation of hemisphere edema in the DM group (Fig. 2). DM animals had 13.1 ___ 2.5% cortical edema (P<0.01) and 7.5 -t- 1.9% hemisphere edema (P<0.01) compared with 55.1 + 9.2% cortical edema and 36.5 ___ 7.2% hemisphere edema in the normal saline controls. Histopathology also confirmed DM's neuroprotective effect with a 92% decrease in cortical IND (DM 3.8 + 0.7%, NS 46.4 -t- 13.3%, P<0.01) (Fig. 2). In non-ischemic rabbits neither DM nor NS animals showed any areas of IND or ischemic edema. Dextromethorphan is an NMDA antagonist that interacts with the NMDA receptor-channel complex, similar to other noncompetitive antagonists including MK-801, phencyclidine (PCP) and ketamine [9, 12]. DM protects against glutamate, N M D A and hypoxic neuronal damage in culture [3, 6] and prevents cerebral injury
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Fig. 2. Dextromethorpban (DM) promotes recoveq¢ of the somatosensory evoked potential (SEP), reduces cortical iscbemic neuronal damage (IND) and decreases cortical edema. Mean ___ S.E.M. % preocclusion amplitude for SEP or % area for I N D and edema. (DM, n = 6 rabbits; NS, n = 5 rabbits). * P < 0 . 0 5 , **P<0.01; M a n n - W h i t n e y U-test.
when administered before ischemia or up to 1 h after the onset of ischemia in animal models [16-19]. Our present study confirms that i.v. DM treatment started 10 rain after ischemia, protects against cortical IND, ischemic edema and promotes electrophysiologic recovery. Previous investigators have suggested that selective blockade of the NMDA receptor underlies the in vivo neuroprotective properties of DM and other NMDA antagonists [17, 18]. However, NMDA antagonists may also favorably alter CBF in such a way to provide protection against ischemia. Tortella et al. showed that DM attenuates post-ischemic hypoperfusion in a rat model of global ischemia [20] and MK-801 is known to significantly affect CBF in various ways depending on the species, anesthetic agent, region of brain examined, and presence or absence of ischemia [13-15]. Our study suggests that DM has a significant influence on rCBF during reperfusion after transient focal cerebral ischemia. Immediately after unocclusion in the NStreated animals, there was a sharp increase in rCBF followed by a gradual deterioration to low levels of flow. In the DM-treated animals, this immediate initial increase was prevented and rCBF recovered in a gradual manner over the course of 4 h of reperfusion. It has been suggested that restoration of blood flow following ischemia may induce reperfusion injury [lt)]. It is possible that the immediate reflow occurring after blood flow is reinstated may be detrimental through oxygen free radical injury or by increasing plasma leakage through a perturbed blood-brain barrier (BBB), thus exacerbating the degree of ischemic edema. It has been demonstrated that moderating the sharp initial rebound in rCBF postischemia reduces cerebral edema in cats subjected to a transient MCA occlusion [10]. In the present study, DM prevented the initial sharp increase in rCBF after transient ischemia. It is, therefore, possible that DM
228 m a y also p r o t e c t against ischemic n e u r o n a l injury by decreasing the degree o f cerebral e d e m a a n d reducing the a m o u n t o f reperfusion injury. I n a d d i t i o n , D M i m p r o v e d the d e l a y e d h y p o p e r f u s i o n in the later stages o f the reperfusion period. Thus, D M m a y also p r o t e c t against ischemic injury by increasing flow to m a r g i n a l l y perfused b r a i n regions d u r i n g reperfusion. The u n d e r l y i n g m e c h a n i s m o f D M ' s influence on r C B F d u r i n g reperfusion has n o t been elucidated. N M D A receptors have n o t been f o u n d on b l o o d vessels [1] a n d N M D A agonists d o n o t affect c e r e b r o v a s c u l a r tone in vitro [7]. W h i l e D M is a v o l t a g e - d e p e n d e n t calcium a n t a g o n i s t [2, 9] a n d c o u l d p o t e n t i a l l y act via this m e c h a n i s m by directly v a s o d i l a t i n g cerebral vessels, we did n o t observe any c h a n g e in r C B F a m o n g non-ischemic a n i m a l s treated with D M over 6 h. O t h e r studies in o u r l a b o r a t o r y using the r a d i o a c t i v e m i c r o s p h e r e technique suggest t h a t D M i m p r o v e s r C B F in the ischemic p e n u m b r a d u r i n g the p e r i o d o f arterial occlusion [11]. Thus, while D M m a y p r i m a r i l y alter C B F in ischemic regions, an alternative h y p o t h e s i s is t h a t these effects are s e c o n d a r y to p r e v e n t i o n o f n e u r o n a l excitotoxicity with preserved a u t o r e g u l a t i o n a n d c o u p l i n g o f b l o o d flow to intact n e u r o n a l m e t a b o l i s m . R e g a r d l e s s o f the m e c h a n ism u n d e r l y i n g D M ' s a l t e r a t i o n o f r C B F d u r i n g ischem i a a n d reperfusion, these f a v o r a b l e changes in C B F m a y serve to p r e v e n t a d d i t i o n a l cerebral injury. W e t h a n k J u t t a Bischof for p r e p a r a t i o n o f the m a n u s cript. This w o r k was s u p p o r t e d by A m e r i c a n H e a r t A s s o c i a t i o n G r a n t - i n - A i d 881069, N I H G r a n t s 1 R O I NS27292-01A2, a n d 2S07 R05353-29, a n d the Valeria K. Bernadt Cerebral Ischemia Fund. 1 Beart, P.M., Sheehan, K.A.M. and Manallack, D.T., Absence of N-methyl-D-aspartate receptors on ovine cerebral microvessels, J. Cereb. Blood Flow Metab., 8 (1988) 879-882. 2 Carpenter, C.L., Marks, S.S., Watson, D.L. and Greenberg, D.A., Dextromethorphan and dextrorphan as calcium channel antagonists, Brain Res., 439 (1988) 372-375. 3 Choi, D.W., Dextrorphan and dextromethorphan attenuate glutamate neurotoxicity, Brain Res., 403 (1987) 333-336. 4 DeLaPaz, R,L., Shibata, D., Steinberg, G.K., Zarnegar, R. and George, C., Acute cerebral ischemia in rabbits: correlation between MR and histopathology, Am. J. Neuroradiol., 12 (1991) 89-95. 5 Dirnagl, U., Kaplan, B., Jacewicz, M. and Pulsinelli, W., Continuous measurement of cerebral cortical blood flow by LaserDoppler flowmetry in a rat stroke model, J. Cereb. Blood Flow Metab., 9 (1989) 589-596.
6 Goldberg, M.P,, Pham, P.-C. and Choi, D.K., Dextrorphan and dextromethorphan attenuate hypoxic injury in neuronal culture, Neurosci. Lett., 80 (1987) 11 15. 7 Hardebo, J.E., Wieloch, T. and Kagstrom, J., Excitatory amino acids and cerebrovascular tone, Acta Physiol. Scand., 136 (1989) 483-485. 8 Isbell, H. and Fraser, H.F., Actions and addiction liabilities ofdromoran derivatives in man, J. Pharmacol. Exp. Ther., 107 (1953) 524-530. 9 Jaffe, D.B., Marks, S.S. and Greenberg, D.A., Action, antagonist drug selectivity for radioligand binding sites on voltage-gated and N-methyl-D-aspartate receptor-gated Ca 2+ channels, Neurosci. Lett., 105 (1989) 227-232. 10 Kuroiwa, T., Shibutani, M. and Okeda, R., Nonhyperemic blood flow restoration and brain edema in experimental focal cerebral ischemia, J. Neurosurg., 70 (1989) 73-80. 11 Lo, E.H. and Steinberg, G.K., Effects of dextromethorphan on regional cerebral blood flow in focal cerebral ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 803-809. 12 Lodge, D., Aram, J.A, Church, J., Davies, S.N., Martin, D, O'Shaughnessy, C.T. and Zeman, S., Excitatory amino acid and phenylcyclidine-like drugs. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Alan R. Liss, New York, 1987, pp. 83-90. 13 Meyer, F.B., Anderson, R.E. and Friedrich, P.F., MK-801 attenuates capillary bed compression and hypoperfusion following incomplete focal cerebral ischemia, J. Cereb. Blood Flow Metab., 10 (1990) 895-902. 14 Nehls, D.G., Park, C.K., MacCormack, A.G. and McCulloch, J., The effects of N-methyl-D-aspartate blockade with MK-801 upon the relationship between cerebral flood flow and glucose utilisation, Brain Res., 511 (1990) 272 279. 15 Park, C.K., Nehls, D.G., Teasdale, G.M. and McCulloch, J., Effect of the NMDA antagonist MK-801 on local cerebral flood flow in focal cerebral ischemia in the rat, J. Cereb. Blood Flow Metab., 9 (1989) 617~522. 16 Prince, D.A. and Feeser, H.R., Dextromethorphan protects against cerebral infarction in a rat model of hypoxia-ischemia, Neurosci. Lett., 85 (1988) 291--296. 17 Steinberg, G.K., Saleh, J. and Kunis, D., Delayed treatment with dextromethorphan and dextrorphan reduces cerebral damage after transient focal ischemia, Neurosci. Lett., 89 (1988) 193-197. 18 Steinberg, G.K., George, C.P., DeLaPaz, R., Shibata, D.K. and Gross, T., Dextromethorphan protects against cerebral injury following transient focal ischemia in rabbits, Stroke, 19 (1988) 1112 1118. 19 Steinberg, G.K., Saleh, J., Kunis, D., DeLaPaz, R. and Zarnegar, S.R., Protective effect of N-methyl-D-asparate antagonists after focal cerebral ischemia in rabbits, Stroke, 20 (1989) 1247-1252. 20 Tortella, F.C., Martin, D.A., Allot, C.P., Steel, J.A., Blackburn, T.P., Loveday, B.E. and Russell, N.J., Dextromethorphan attenuates post-ischemic hypoperfusion following incomplete global ischemia in the anesthetized rat, Brain Res., 482 (1989) 179-183.
225
© 1991 ElsevierScientific Publishers Ireland Ltd. All rights reserved0304-3940/91/$ 03.50 NSL08233
Dextromethorphan alters cerebral blood flow and protects against cerebral injury following focal ischemia G a r y K . Steinberg, E n g H . L o , D a v i d M. K u n i s , G e r a l d A. G r a n t , Alex P o l j a k a n d R o b e r t D e L a P a z Department of Neurosurgery and Division of Neuroradiology, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.)
(Received 28 May 1991; Revisedversion received 20 August 1991; Accepted 28 August 1991) Key words: Dextromethorphan; Cerebral blood flow; Cerebral ischemia; N-Methyl-D-aspartate;Glutamate
The effectsof the N-methyl-o-aspartate (NMDA) antagonist dextromethorphan (DM) on regional cerebral blood flow (rCBF) and cerebral injury were studied in a rabbit model of transient focal ischemia. Anesthetized rabbits underwent 2 h occlusion of the left internal carotid, middle cerebral and anterior cerebral artery, followedby 4 h of reperfusion. Ten minutes after the onset of ischemia they were treated with either i.v. DM 20 mg/kg followed by 10 mg/kg/h (n =6) or normal saline (NS, n = 5). Control rabbits received DM (n = 3) or NS (n = 2) infusion without arterial occlusion. DM attenuated the sharp, post-ischemicrise in rCBF seen during reperfusion within the ischemiccore of NS controls (DM 31 ~; pre-ischemicvalue, NS 92%). DM also improved the delayed post-ischemic hypoperfusion compared with controls. DM infusion without arterial occlusion did not change rCBF values. Compared with NS controls, DM treated animals demonstrated recovery of the somatosensory evoked potential (DM 96% pre-ischemic values, NS 24%), 76% reduction in cortical edema and 92% decrease in cortical ischemic neuronal damage. We conclude that DM's effect on CBF may contribute to its neuroprotective action.
Dextromethorphan (D- 3-methoxy-N-methylmorphinan; D M ) is an opioid compound, but devoid of most narcotic properties [8]. D M is a noncompetitive, Nmethyl-D-aspartate ( N M D A ) antagonist [9, 12] that attenuates glutamate toxicity, N M D A - i n d u c e d damage and hypoxic injury in neuronal culture [3, 6]. D M also prevents ischemic neuronal damage and edema in animal models of focal cerebral ischemia when administered before ischemia or in a delayed manner after ischemia [1619]. It has been postulated that the neuroprotective effects of D M and other N M D A antagonists are related to blockade of neuronal N M D A receptors [17, 18]. However, it is possible that D M and these other N M D A antagonists provide neuroprotection by altering cerebral blood flow or metabolism. We undertook this study to investigate the effects of D M on regional cerebral blood flow (rCBF) and cerebral injury in a rabbit model of transient focal i schemia [17-19]. Male New Zealand white rabbits (2.5-3.5 kg) were anesthetized with 3 % halothane delivered by mask. After catheterization of the femoral artery and ear vein, the rabbits were tracheostomized, immobilized with 0.2 mg/ kg i.v. pancuronium bromide and artificially ventilated. Correspondence: G.K. Steinberg, Department of Neurosurgery S006, Stanford University Medical Center, Stanford, CA 94305, U.S.A.
Anesthesia was maintained with 0.5% halothane in 0.5 l/min 02:4.5 l/min air. Mean arterial pressure (MAP), end-expired CO2, heart rate, rectal temperature and temporalis muscle temperature were measured continuously. Arterial blood gasses, hematocrit and blood glucose were monitored intermittently. Rectal temperature was controlled at 39°C and temporalis muscle temperature at 37.5°C using a heating lamp and heating pad. Endexpired CO2 was maintained at 30-35 m m Hg by adjusting the ventilation rate. Arterial base deficit was corrected as necessary with i.v. sodium bicarbonate. After immobilization of the rabbit head in a stereotactic frame, the dorsal scalp was removed and somatosensory evoked potential (SEP) bone-screw electrodes were inserted 5 m m lateral (left and right) to bregma. Stimulating electrodes were placed over left and right median nerves. Square-wave stimuli (10 mA, 0.25 ms) were delivered at 4.1 Hz and SEPs were recorded with a Nicolet Pathfinder II electrodiagnostic system. One hundred and fifty responses were averaged for each measurement. SEP amplitudes were measured from the peak of the first major positive deflection to the trough of the adjacent negative deflection [18]. F o r analysis, SEP amplitudes were expressed as percentages of pre-ischemic values. To measure rCBF, two windows (2 m m diameter) were drilled over the left hemisphere: the first 15 m m anterior and 15 m m lateral to bregma, the second 0 m m anterior
226
and l0 mm lateral to bregma. The dura was left intact to prevent injury to the brain. A saline drip was also used during drilling to prevent overheating of the underlying brain. Laser-Doppler flow probes (TSI BPM 403A) were placed over the rCBF windows and using a micromanipulator were advanced under microscopic guidance to the surface of the brain. Care was taken not to damage the brain while adjusting the probe position. Since the laser-Doppler method has been demonstrated to provide accurate measurements of rCBF changes, but not absolute flow [5], all measurements of rCBF in this study were expressed as relative percentages of pre-ischemic or pre-drug values. In order to insure a uniform degree of pretreatment ischemia in all rabbits, we only used animals in which the SEPs were completely abolished within 10 min of arterial occlusion. Eleven rabbits underwent transorbital clip occlusion of the left internal carotid, proximal left anterior cerebral and proximal left middle cerebral arteries for 2 h, followed by 4 h of reperfusion. During the 2 h occlusion period, the MAP was kept 60-70 mm Hg by adjusting the halothane concentration between 0.5-0.7%. A loading dose of DM (20 mg/kg) followed by maintenance doses of 10 mg/kg/h (n=6) or an equivalent volume of normal saline (NS; n = 5) was blindly administered starting 10 min after occlusion. In order to examine DM's effects on rCBF in the non-ischemic brain, 5 anesthetized rabbits were administered i.v. DM (n = 3) or NS (n=2) without undergoing arterial occlusion. The dose of DM and experimental techniques were otherwise identical to those for the ischemic rabbits. Rabbits were sacrificed with sodium pentobarbital and perfused with saline followed by 10% formalin [17-19]. The brains were kept in the skull overnight, then removed and stored in formalin. Magnetic resonance imaging (MRI) studies were performed using sequences described previously (i .5 Tesla General Electric Signal Systems, 3 mm slice thickness, TR 2500, TE 100) [19]. Using 5 standard coronal images, abnormal high intensity areas were expressed as a percentage of total cortical or hemisphere area. Previous studies have shown that regions of abnormal MRI signal represent areas ofcytotoxic and vasogenic edema [4]. Histological examination of 5 standard coronal, hematoxylin-eosin stained sections (paraffin embedded, 6 #m thick) were performed to measure areas of ischemic neuronal damage (IND) [19]. An average percentage area ischemic edema or IND for each animal was calculated as the total area of edema or IND for all 5 coronal levels divided by the total area of cortex or hemisphere for all 5 levels. Statistical analyses utilized non-parametric Mann-Whitney U-tests and Wilcoxon sign tests; P < 0.05 was considered significant. There were no differences between groups in any syste-
mic parameters before arterial occlusion, during arterial occlusion or during reperfusion. These included rectal and temporalis muscle temperature, MAP, heart rate, paO2, paC02, pH, hematocrit, glucose, halothane administered, or volume of i.v. fluid infused. Laser-Doppler measurements demonstrated an immediate drop in rCBF after occlusion, however, not all rCBF windows showed similar degrees of reduced rCBF, demonstrating the variable distribution of the ischemic lesion. For this analysis, only sites where rCBF decreased below 20 % of pre-ischemic values (ischemic core) were used. In the DM group, one rabbit showed a de-
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Fig. I. Regional cerebral blood flow (rCBF) in dextromethorphan (DM) or normal saline (NS) treated rabbits. A: after transient focal ischemia, dextromethorphan prevents the post-ischemic rise in rCBF with the onset of reperfusion. D M also improves the delayed postisehemic hypoperfusion observed in the NS group. N u m b e r of rCBF window measurements at each time point (DM, n = 7; NS, n = 7). B: in non-ischemic rabbits rCBF does not differ between D M and NS groups (DM, n = 6 rCBF windows; NS, n = 4 rCBF windows). Mean _+ S.E.M. * P < 0 . 0 5 M a n n Whitney U-test for comparison between DM and NS. t P < 0 . 0 5 Wilcoxon signed rank test for comparison with pre-occlusion rCBF values.
227
crease in rCBF below 20~ in both windows and 5 rabbits showed decreases in only one window. In the NS group, two rabbits showed decreases below 20 % in both windows and 3 rabbits showed decreases below 20% in only one window. During the 2 h occlusion period, rCBF remained severely depressed, and no significant differences were found between the DM and NS groups (Fig. 1A). However, significant differences in rCBF patterns were observed during the reperfusion period. The NS group demonstrated an immediate, sharp post-ischemic rise in rCBF (92% of pre-ischemic values at 0.5 h of reperfusion), followed by a gradual deterioration to 35 % pre-ischemic values at 4 h of reperfusion. In the DM group, the immediate rise in rCBF after unocclusion was eliminated (P<0.05 at 0.5 h of reperfusion compared with NS controls), and rCBF gradually recovered to preischemic values at 4 h of reperfusion (Fig. 1A). In the NS group, rCBF values during the later stages of reperfusion (5 and 5.5 h) were significantly lower than preocclusion values ( P < 0.05, Wilcoxon signed rank test). rCBF in the DM group at these time points was not significantly different from pre-occlusion values. In the non-ischemic rabbits, rCBF declined slightly over the 6 h experiment, but did not differ between DM and NS groups (Fig. 1B). SEP amplitudes were reduced to zero between 5 and 10 min after onset of ischemia in all rabbits within both DM and NS groups. After reperfusion, SEP amplitudes in the DM group showed greater recovery than the NS group (Fig. 2). In the DM group, SEP recovered to 96.3 ___ 21.3% (mean -t- S.E.M.) of pre-ischemic values, whereas in the NS groups, SEP amplitudes were 23.8 + 16.9 % of pre-ischemic values (P < 0.05). In non-ischemic rabbits, SEP amplitudes did not change significantly with either DM or NS treatment. MRI analysis demonstrated 76% reduction in cortical ischemic edema and 79 % attenuation of hemisphere edema in the DM group (Fig. 2). DM animals had 13.1 ___ 2.5% cortical edema (P<0.01) and 7.5 -t- 1.9% hemisphere edema (P<0.01) compared with 55.1 + 9.2% cortical edema and 36.5 ___ 7.2% hemisphere edema in the normal saline controls. Histopathology also confirmed DM's neuroprotective effect with a 92% decrease in cortical IND (DM 3.8 + 0.7%, NS 46.4 -t- 13.3%, P<0.01) (Fig. 2). In non-ischemic rabbits neither DM nor NS animals showed any areas of IND or ischemic edema. Dextromethorphan is an NMDA antagonist that interacts with the NMDA receptor-channel complex, similar to other noncompetitive antagonists including MK-801, phencyclidine (PCP) and ketamine [9, 12]. DM protects against glutamate, N M D A and hypoxic neuronal damage in culture [3, 6] and prevents cerebral injury
~.~JNormal Saline I l l Dextrornethorphan 100
.o 0 E5
~ i a_
75
~_
~o 2 E a
25
**
0
--
SEP
Cortical I~!g
Cortical Edema
Fig. 2. Dextromethorpban (DM) promotes recoveq¢ of the somatosensory evoked potential (SEP), reduces cortical iscbemic neuronal damage (IND) and decreases cortical edema. Mean ___ S.E.M. % preocclusion amplitude for SEP or % area for I N D and edema. (DM, n = 6 rabbits; NS, n = 5 rabbits). * P < 0 . 0 5 , **P<0.01; M a n n - W h i t n e y U-test.
when administered before ischemia or up to 1 h after the onset of ischemia in animal models [16-19]. Our present study confirms that i.v. DM treatment started 10 rain after ischemia, protects against cortical IND, ischemic edema and promotes electrophysiologic recovery. Previous investigators have suggested that selective blockade of the NMDA receptor underlies the in vivo neuroprotective properties of DM and other NMDA antagonists [17, 18]. However, NMDA antagonists may also favorably alter CBF in such a way to provide protection against ischemia. Tortella et al. showed that DM attenuates post-ischemic hypoperfusion in a rat model of global ischemia [20] and MK-801 is known to significantly affect CBF in various ways depending on the species, anesthetic agent, region of brain examined, and presence or absence of ischemia [13-15]. Our study suggests that DM has a significant influence on rCBF during reperfusion after transient focal cerebral ischemia. Immediately after unocclusion in the NStreated animals, there was a sharp increase in rCBF followed by a gradual deterioration to low levels of flow. In the DM-treated animals, this immediate initial increase was prevented and rCBF recovered in a gradual manner over the course of 4 h of reperfusion. It has been suggested that restoration of blood flow following ischemia may induce reperfusion injury [lt)]. It is possible that the immediate reflow occurring after blood flow is reinstated may be detrimental through oxygen free radical injury or by increasing plasma leakage through a perturbed blood-brain barrier (BBB), thus exacerbating the degree of ischemic edema. It has been demonstrated that moderating the sharp initial rebound in rCBF postischemia reduces cerebral edema in cats subjected to a transient MCA occlusion [10]. In the present study, DM prevented the initial sharp increase in rCBF after transient ischemia. It is, therefore, possible that DM
228 m a y also p r o t e c t against ischemic n e u r o n a l injury by decreasing the degree o f cerebral e d e m a a n d reducing the a m o u n t o f reperfusion injury. I n a d d i t i o n , D M i m p r o v e d the d e l a y e d h y p o p e r f u s i o n in the later stages o f the reperfusion period. Thus, D M m a y also p r o t e c t against ischemic injury by increasing flow to m a r g i n a l l y perfused b r a i n regions d u r i n g reperfusion. The u n d e r l y i n g m e c h a n i s m o f D M ' s influence on r C B F d u r i n g reperfusion has n o t been elucidated. N M D A receptors have n o t been f o u n d on b l o o d vessels [1] a n d N M D A agonists d o n o t affect c e r e b r o v a s c u l a r tone in vitro [7]. W h i l e D M is a v o l t a g e - d e p e n d e n t calcium a n t a g o n i s t [2, 9] a n d c o u l d p o t e n t i a l l y act via this m e c h a n i s m by directly v a s o d i l a t i n g cerebral vessels, we did n o t observe any c h a n g e in r C B F a m o n g non-ischemic a n i m a l s treated with D M over 6 h. O t h e r studies in o u r l a b o r a t o r y using the r a d i o a c t i v e m i c r o s p h e r e technique suggest t h a t D M i m p r o v e s r C B F in the ischemic p e n u m b r a d u r i n g the p e r i o d o f arterial occlusion [11]. Thus, while D M m a y p r i m a r i l y alter C B F in ischemic regions, an alternative h y p o t h e s i s is t h a t these effects are s e c o n d a r y to p r e v e n t i o n o f n e u r o n a l excitotoxicity with preserved a u t o r e g u l a t i o n a n d c o u p l i n g o f b l o o d flow to intact n e u r o n a l m e t a b o l i s m . R e g a r d l e s s o f the m e c h a n ism u n d e r l y i n g D M ' s a l t e r a t i o n o f r C B F d u r i n g ischem i a a n d reperfusion, these f a v o r a b l e changes in C B F m a y serve to p r e v e n t a d d i t i o n a l cerebral injury. W e t h a n k J u t t a Bischof for p r e p a r a t i o n o f the m a n u s cript. This w o r k was s u p p o r t e d by A m e r i c a n H e a r t A s s o c i a t i o n G r a n t - i n - A i d 881069, N I H G r a n t s 1 R O I NS27292-01A2, a n d 2S07 R05353-29, a n d the Valeria K. Bernadt Cerebral Ischemia Fund. 1 Beart, P.M., Sheehan, K.A.M. and Manallack, D.T., Absence of N-methyl-D-aspartate receptors on ovine cerebral microvessels, J. Cereb. Blood Flow Metab., 8 (1988) 879-882. 2 Carpenter, C.L., Marks, S.S., Watson, D.L. and Greenberg, D.A., Dextromethorphan and dextrorphan as calcium channel antagonists, Brain Res., 439 (1988) 372-375. 3 Choi, D.W., Dextrorphan and dextromethorphan attenuate glutamate neurotoxicity, Brain Res., 403 (1987) 333-336. 4 DeLaPaz, R,L., Shibata, D., Steinberg, G.K., Zarnegar, R. and George, C., Acute cerebral ischemia in rabbits: correlation between MR and histopathology, Am. J. Neuroradiol., 12 (1991) 89-95. 5 Dirnagl, U., Kaplan, B., Jacewicz, M. and Pulsinelli, W., Continuous measurement of cerebral cortical blood flow by LaserDoppler flowmetry in a rat stroke model, J. Cereb. Blood Flow Metab., 9 (1989) 589-596.
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