Spreading Depression

Journal of Cerebral Blood Flow and Metabolism 17:586-590 1997 The International Society of Cerebral Blood Flow and Metabolism Published by Lippincott-Raven Publishers, Philadelphia In Vivo Uptake of eh]nimodipine into Brain During Cortical Spreading Depression Sachiko Osuga, Antoine M. Hakim, Hitoshi Osuga, and Matthew J. Hogan Neuroscience Research Institute, University of Ottawa, Ottawa, Canada Summary: We report autoradiographic measurements of the in vivo uptake of ehjnimodipine during the nonischemic depolarization of cortical spreading depression (CSD) in rat brain. [3HJNimodipine uptake in brain was determined regionally in rats undergoing CSD (n = 8) and was significantly increased in cortex (14 ± 7%) and hippocampus (10 ± 6%) on the stimulated side relative to the contralateral hemisphere when compared with the same measurements in a control group (n = 8). A similar measurement using the physiologically inert radiotracer e4cj iodoantipyrine to control for potential effects of CSD on radioligand distribution showed a minimal increase (2.4 ± 0.7%) of radiotracer uptake in cortex after CSD. This increase was significantly less than that observed in the [3HJnimodipine uptake studies. We hypothesize that increased in vivo ehjnimodipine uptake in CSD identifies regions of depolarization and thus infers activation of the L-type voltage sensitive calcium channels. Key Words: Cortical spreading depression-voltage sensitive calcium channels-nimodipineautoradiography. Cortical spreading depression (CSD) is a reversible wave of depressed electrocortical activity and direct current (DC) depolarization that propagates across the cerebral cortex at a characteristic rate of 3 mm/min (Leao, 1944). The brief period of brain tissue depolarization is associated with massive shifts of K+ out of cells and Na+, CC Ca2+ and water into cells (see review by Somjen et ai., 1992). Excitatory neurotransmission and calcium channel activation may be factors in CSD. Cortical spreading depression is reduced or completely suppressed with N-methyl-D-aspartate receptor antagonists (Lauritzen and Hansen, 1992; McLachlan, 1992) and blockade of voltage-sensitive calcium channels (VSCC) prevents K+ induced spreading depression in hippocampal slice preparations (Jing et ai., 1993). We have previously shown increased in vivo binding of the 1 A-dihydropyridine L-type VSCC antagonist eh]nimodipine to the L-type VSCC in acutely ischemic Received February 10, 1995; final revision received November 27, 1996; accepted December 27, 1996. This work was supported by the Medical Research Council of Canada, the Heart and Stroke Foundation of Ontario, and Miles Pharmaceutical, Inc., Etobicoke, Ontario, Canada. Address correspondence and reprint requests to Dr. M. Hogan, Neuroscience Research Institute, University of Ottawa, 451 Smyth Road, Ottawa, Ontario KIH 8M5 CANADA. Abbreviations used: CSD, cortical spreading depression; CBF, cerebral blood flow; DC, direct current; VSCC, voltage-sensitive calcium channel. brain by using quantitative autoradiography (Hakim and Hogan, 1991; Hogan et ai., 1991). We proposed that increased in vivo uptake of eh]nimodipine into ischemic brain was a consequence of the greater affinity of the L-type VSCC in depolarized cell membranes to bind l,4-dihydropyridine VSCC antagonists (Kokubun et ai., 1986; Triggle and Rampe, 1989) thus inferring activation of this channel. If this hypothesis is correct in vivo uptake of eh]nimodipine should increase during the nonischemic depolarization associated with CSD. METHODS Adult male Sprague Dawley rats were fasted overnight before study. Halothane anesthesia (4% induction, 0.5% maintenance) was maintained throughout surgery and during recurrent CSD. eh]nimodipine binding during cortical spreading depression Mean arterial blood pressure and arterial blood gases were monitored in spontaneously breathing rats. Body temperature was maintained between 36 and 37 C. Burr holes were placed stereotactically over the right occipital and right frontal cortices. A microdialysis probe was inserted 2 mm into the right occipital cortex. To record CSD, a 13 /.1m-diameter, platinum wire electrode was inserted 2 mm into the right frontal cortex and connected to a DC amplifier (Gould Instruments Inc., Valley View, OH, U.S.A.). In 8 rats CSD was induced by infusion of 3 mol/l KCI solution through the microdialysis probe at a starting rate of 2 /.1L!min. Control animals (n = 8) were infused 586

NIMODIPlNE UPTAKE IN SPREADING DEPRESSION 587 with nonnal saline at the same rate. After 60 minutes of recurrent CSD, 200 j.lci of ehjnimodipine (specific activity 130 Ci/mmol, NEN-Dupont, Boston, MA, U.S.A.) in 600 j.ll of carrier (BAY e9736 placebo, Miles Phannaceuticals, Etobicoke, Ontario, Canada) were infused intravenously over 3 minutes at the start of the next recorded DC potential shift. In control studies the radiotracer was infused after 60 minutes of nonnal saline perfusion of the microdialysis probe. After 15 minutes of radiotracer circulation plasma, ehjnimodipine activity was measured, the rat was decapitated, and the brain was immediately removed and frozen at -70 C. e4c]iodoantipyrine uptake during cortical spreading depression ehjnimodipine freely crosses the blood brain barrier (Van den Kerckhoff and Drewes, 1985) and its initial clearance may be influenced by cerebral blood flow (CBF) changes during CSD. To assess for this possible effect, the uptake of the physiological inert and blood brain barrier penneable radiotracer e4c]jodoantipyrine (Sakurada et ai., 1978) was measured. Probe placements were identical to the ehjnimodipine studies. After 60 minutes of either recurrent CSD (n = 6) or saline perfusion (n = 6) 30 j.lci of e4c]iodoantipyrine (specific activity 57 mci/mmol, Amersham International PLC, England) in 600 j.ll of nonnal saline were infused over 3 minutes at the start of the next recorded CSD. After 15 minutes of radiotracer circulation plasma e4c]iodoantipyrine activity was measured, the rat was decapitated, and the brain was rapidly removed and frozen at -70 C. To better control arterial Pco2 and associated CBF changes, these rats were mechanically ventilated with 30% O2 to 70% N20 in addition to halothane. Autoradiography Horizontal cryostat brain sections 20-j.Lm thick were obtained every 140 j.lm and apposed to tritium sensitive film (Hyperfilm, Amersham, Oakville, Ontario, Canada) for ehjnimodi pine studies or Kodak SB-5 film (Picker International, Brampton, Ontario, Canada) for [14C]iodoantipyrine studies. Tritium autoradiographs were calibrated using tissue paste standards prepared in the laboratory from forebrain homogenates. Microscale standards (Amersham International PLC, England) were used to calibrate the 14C autoradiographs. Autoradiographs were digitized (Imaging Research Inc., St. Catharines, Ontario, Canada) and regional radiotracer concentrations detennined using a standardized region of interest template at five horizontal levels (Fig. 1). The observer was blinded to the treatment status of the rats. Previously determined metabolite corrections (Hogan et ai., 199 I) were applied FIG. 1. Region of interest template used to analyze the autoradiographic binding data in one cerebral hemisphere. The five groups of regions of interest which form the volumes of interest, generated by anatomical and cluster analysis criteria, are identified by the hatched markings. J Cereb Blood Flow Metab. Vol. 17, No.5, 1997

588 S. OSUGA ET AL. and [3HJnimodipine volume of distribution calculated as the ratio of tissue [3HJnimodipine over plasma [3HJnimodipine concentration (Lassen and Perl, 1979). Data analysis and statistics Physiological data comparisons were performed using independent t tests. The ratio of the volume of distribution of ehlnimodipine in the stimulated hemisphere over that in the contralateral hemisphere was calculated for each region of interest. Striatal and hippocampal regions of interest data were grouped and K-means cluster analysis (Hartigan and Wong, 1979) was used 'to subdivide the cortical regions of interest into three groups. All subsequent analysis used data averaged over these five volumes of interest (Fig. 1). The Mann-Whitney U test (BMDP Statistical Software, Inc., Los Angeles, CA, U.S.A.) with Bonferonni correction was used to assess differences between the CSD and control groups. RESULTS Physiologic measurements Mean arterial blood pressure and arterial blood gases did not differ between the CSD and control groups in the [ 3 H]nimodipine and C 4 C]iodoantipyrine studies. Peo2 was lower and better controlled in the e 4 C]iodoantipyrine studies. Cortical spreading depression Recurrent DC potential shifts with initial negative deflection of 1 to 5 m V of 30 to 60 seconds duration consistent with CSD depolarization (Somjen et ai., 1992; McLachlan, 1992) were recorded from the frontal cortex. In the eh]nimodipine CSD group, 8.0 ± 2.6 DC depo- FIG 2. Autoradiographs of four horizontal brain sections from a selected study with recurrent CSD showing in vivo [3Hlnimodipine uptake. The stimulated hemisphere is on the left side of the sections. The figure has been contrast enhanced to show the increase in [3Hlnimodipine uptake in the frontal cortex ipsilateral to the recurrent CSD. Increased activity was also evident in the ipsilateral hippocampus but no change in uptake occurred in striatum. The location of the microdialysis probe and surrounding narrow band of depolarization is indicated by the asterisk. The arrow by the top left section indicates the location of the recording electrode. The horizontal sections correspond to the first four levels in Fig. 1. The pseudocolor scale has lower activities in blue and higher activities in yellow and red. J Cereh Blood Flow Metab. Vol. 17, No.5, 1997

NIMODIPINE UPTAKE IN SPREADING DEPRESSION 589 larizations were recorded whereas in the e 4 C]iodoantipyrine CSD group, 9.0 ± 2.2 DC depolarizations were observed. In the r 3 H]nimodipine studies only one DC depolarization was observed after eh]nimodipine infusion whereas in the C 4 C]iodoantipyrine studies six DC depolarizations were observed in five rats after radiotracer infusion. One DC depolarization was observed in the [ 3 H]nimodipine control group. eh]nimodipine binding Figure 2 shows autoradiographs of in vivo uptake of eh]nimodipine into brain in four horizontal sections (corresponding to the first four levels in Fig. 1) from a selected study in the recurrent CSD group. The stimulated hemisphere is on the left side of these images. [ 3 H]Nimodipine uptake was increased in both cortex and hippocampus of the CSD-stimulated hemisphere relative to the contralateral hemisphere. No change was observed in striatum. The narrow margin of intense eh]nimodipine uptake around the microdialysis probe was excluded from analysis. The ratio of eh]nimodipine uptake in the stimulated hemisphere over the contralateral hemisphere for each volume of interest is summarized in Table 1. This ratio was increased in cortex I by 14% (p = 0.019), in cortex 2 by 11 % (p = 0.031), and in the hippocampus by lo% (p = 0.016) in the CSD group compared with the control group. In the control group, the average cortical eh]nimodipine volume of distribution was lo4 ± 20 ml/i00 g and was 112 ± 17 ml/loo g in the striatum and 125 ± 23 ml/i00 g in the hippocampus. Volumes of distribution in the contralateral hemisphere did not differ between control and CSD studies. e4c]iodoantipyrine uptake Ratios of e 4 C]iodoantipyrine uptake in the stimulated hemisphere over the contralateral hemisphere for each volume of interest ranged from 1.005 ± 0.015 to 1.035 ± 0.027 in the control group and from 1.031 ± 0.006 to 1.065 ± 0.042 in the CSD group. This ratio was only TABLE 1. Ratio of { 3 HJnimodipine uptake in volumes of interest Region Cortex-I Cortex-2 Cortex-3 Striatum Hippocampus Controls (n = 8) 1.00 ± 0.02 0.97 ± 0.04 0.98 ± 0.04 0.99 ±0.02 0.96 ± 0.03 r'hlnimodipine Uptake Ratio Spreading Depression (n = 8) 1.l4 ± 0.07a 1.08 ± 0.09b 1.04 ± 0.07 0.99 ± 0.05 1.06 ± 0.06c Ratio of l'hlnimodipine volumes of distribution in volumes of interest in the hemisphere with probe implantation over the corresponding volume of interest in the contralateral hemisphere for both control and cortical spreading depression groups. Differs from controls at ap = 0.019. bp = 0.031, cp = 0.016 (Mann-Whitney U [two tailedl with Bonferonni correction for multiple comparison). increased in cortex 1 by 2.4% (p = 0.032) in the CSD group compared with the control group. This increased uptake of e 4 C]iodoantipyrine within cortex 1 was significantly less than the increase observed for eh]nimodipine uptake within the same region (p = 0.02). DISCUSSION We have shown increased in vivo eh]nimodipine uptake in the cortex and hippocampus ipsilateral to recurrent CSD in rat brain. Only a minimal increase in the cortex was observed with the physiologically inert radiotracer [ 14 C]iodoantipyrine administered in an identical manner. We hypothesize that increased eh]nimodipine uptake is a consequence of cell-membrane depolarization indicating activation of the L-type VSCC during CSD. We have previously shown increased in vivo uptake of eh]nimodipine in ischemic brain (Hakim and Hogan, 1991) with kinetics of in vivo binding consistent with saturable and specific binding to the L-type VSCC (Hogan et al, 1991). This increased uptake is rapidly reversible with restoration of CBF (Hogan and Hakim, 1992; Takizawa et ai, 1994) and we concluded that in vivo eh]nimodipine binding may be used to identify transient cell-membrane depolarization and thus infer activation of the L-type VSCc. The current study extends these observations to a model of nonischemic depolarization in which interpretation of binding changes is not confounded by severe CBF depression and histologic injury. The 14% increase of eh]nimodipine uptake during CSD is less than the 90% increase observed in irreversible focal cerebral ischemia (Hakim and Hogan, 1991). In ischemia, tissue depolarization is continuous whereas CSD produces an intense but transient depolarization. In vivo binding of eh]nimodipine to the L-type VSCC may require up to 30 minutes to reach equilibrium (Hogan et ai., 1991). Binding to the L-type VSCC is reversible with dissociation occurring over minutes in membrane fraction preparations (Glossmann and Ferry, 1985). The current experimental design was a compromise between attaining equilibrium of distribution of radiotracer between plasma and brain but preventing complete dissociation of [ 3 H]nimodipine bound to the L-type VSCC after repolarization. Only a fraction of the available binding sites during depolarization may be expected to remain labelled at study termination and our observations will underestimate the degree of depolarization during CSD. Cerebral blood flow may increase by 100% during the initial depolarization of CSD (Hansen et ai., 1980) but subsequently CBF decreases to approximately 70% of control values (Lauritzen et ai., 1982; Duckrow, 1991). The in vivo uptake of [ 14 C]iodoantipyrine administered in an identical manner to eh]nimodipine showed only a small increase in cortical uptake after CSD. This increase J Cereb Blood Flow Metab, Vol. 17, No. 5, 1997

590 S. OSUGA ET AL. was significantly less than the more widespread increase observed with eh]nimodipine despite a greater number of CSD depolarizations after radiotracer administration in the [ 14 C]iodoantipyrine studies. We have previously observed that eh]nimodipine distribution between brain and plasma was not impaired even with marked CBF reduction (Hogan et al 1991). Thus increased r 3 H]nimodipine uptake 15 minutes after CSD is not a consequence of CBF fluctuations. The blood volume in brain has been estimated to be 3.4 to 4.5 ml/i00 g for the range of arterial Pc02 values observed in these studies (Schockley and LaManna, 1988). The 14 ml/loo g increase in eh]nimodipine uptake following CSD cannot be attributed to changes in this volume. Tissue injury may alter nonspecific binding of eh]nimodipine (Hogan et ai, 1991) but pathological changes are not observed in CSD (Nedergaard and Hansen, 1988). Thus we hypothesize that the increased uptake of eh]nimodipine observed in this study is a consequence of transient depolarization during CSD and is an indirect indication of L-type VSCC activation. In cerebral ischemia, CSD may be harmful by increasing the energy demands of tissue in the peri-infarct border zone (Mies et al., 1993). Ischemia-induced CSD may explain the small increase in eh]nimodipine uptake previously report4(l (Hakim and Hogan, 1991) in the cingulate cortex adjacent to ischemic brain in a rat model of middle cerebral artery occlusion. It has also been observed that CSD may induce a delayed neuroprotection to subsequent ischemia (Kawahara et al; 1994; Matsushima et al., 1996). Both CSD and calcium-channel activation will induce immediate early gene expression (Herrera and Robertson, 1990, Murphy et al., 1991) and may underlie the molecular changes observed in CSD. Thus, the phenomenon of CSD with its nonischemic activation of calcium channels may be a useful experimental tool to further probe the metabolic and molecular responses involved in neuronal survival in neurological disease. Acknowledgment: We thank Dr. Kazushi Matsushima for his technical assistance and Ms. Georgette Roy and Ms. Rose Moore their secretarial assistance. REFERENCES Duckrow RB (1991) Regional cerebral blood flow during spreading cortical depression in conscious rats. J Cereb Blood Flow Metab 11:150--154 Glossmann H, Ferry DR (1985) Assay for calcium channels. Methods EnzymoI109:513-550 Hakim AM, Hogan MJ (1991) In vivo binding of nimodipine in the brain: I. The effect of focal cerebral ischemia. J Cereb Blood Flow Metab 11:762-770 Hansen AJ, Quistorff B, Gjedde A (1980) Relationship between local changes in cortical blood flow and extracellular K+ during spreading depression. Acta Physiol Scand 109: 1--6 Hartigan JA, Wong MA (1979) A K-means clustering algorithm: algorithm 136. Appl Statist 28: 100--108 Herrera DG, Robertson HA (1990) Application of potassium chloride to the brain surface induces the c-fos proto-oncogene: reversal by MK-801. Brain Res 510:166--170 Hogan MJ, Gjedde A, Hakim AM (1991) In vivo binding of nimodipine in the brain: II. Binding kinetics in focal cerebral ischemia. J Cereb Blood Flow Metab 11 :771-778 Hogan MJ, Hakim AM (1992) Reversibility of nimodipine binding to brain in transient cerebral ischemia. J Neurochem 59: 1745-1752 Jing J, Aitken PG, Somjen GC (1993) Role of calcium channels in spreading depression in rat hippocampal slices. Brain Res 604: 251-259 Kawahara N, Reutzler CA, Klatzo I (1994) A delayed effect of spreading depression on brain tissue associated with protection against ischemic neuronal damage. Brain Path 4:509 Kokubun S, Prod' hom B, Becker C, Porzig H, Reuter H (1986) Studies on Ca channels in intact cardiac cells: voltage dependent effects and cooperative interactions of dihydropyridine enantiomers. Mol Pharmacal 30:571-584 Lassen NA, Perl W (1979) Tracer Kinetic Methods in Medical Physiology. New York, Raven Press, Lauritzen M, J0rgensen MB, Diemer NH, Gjedde A, Hansen AJ (1982) Persistent oligemia of rat cerebral cortex in the wake of spreading depression. Ann Neurol 12:469-474 Leao AAP (1944) Spreading depression of activity in the cerebral cortex. J Neurophysiol 7:359-390 Matsushima K, Hogan MJ, Hakim AM (1996) Cortical spreading depression protects against subsequent focal cerebral ischemia in rats. J Cereb Blood Flow Metab 16:221-226 McLachlan RS (1992) Suppression of spreading depression of Leao in neocortex by an N-methyl-D-aspartate receptor antagonist. Can J Neurol Sci 19:487-491 Mies G, Iijima T, Hossmann K-A (1993) Correlation between periinfarct DC shifts and ischemic neuronal damage in rat. New'oreport 4:709-711 Murphy TH, Worley PF, Baraban JM (1991) L-type voltage-sensitive calcium channels mediate synaptic activation of immediate early genes. Neuron 7:625-635 Nedergaard M, Hansen AJ (1988) Spreading depression is not associated with neuronal injury in the normal brain. Brain Res 449:395-398 Sakurada 0, Kennedy C, Jehle J, Brown JD, Carbin L, Sokoloff L (1978) Measurement of local cerebral blood flow with iodo[14c]antipyrine. Am J Physiol 234:H59-H66 Shockley RP, LaManna JC (1988) Determination of rat cerebral cortical blood volume changes by capillary mean transit time analysis during hypoxia, hypercapnia and hyperventilation. Brain Res 454: 170--178 Somjen GG, Aitken PG, Cz6h GL, Herreras 0, Jing J, Young IN (1992) Mechanisms of spreading depression: a review of recent findings and a hypothesis. Can J Physiol Pharmacol 70:S248-S254 Takizawa S, Hogan MJ, Buchan A, Hakim AM (1994) In vivo binding of [,H]nimodipine in rat brain after transient forebrain ischemia. J Cereb Blood Flow Metab 14:397-405 Triggle DJ, Rampe D (1989) l,4-dihydropyridine activators and antagonists: structural and functional distinctions. TIPS 10:507-511 Van den Kerckhoff W, Drewes LR (1985) Transfer of the Caantagonists nifedipine and nimodipine across the blood-brain barrier and their regional distribution in vivo. J Cereb Blood Flow Metab 5(suppl I):S459-S460 J Cereb Blood Flow Metab, Vol. 17, No.5, 1997