C O R R E L ATES OF EXPERIMENTAL BRAIN ISCHEMIA: QUANTIFIED EEG ANALY S I S

C O R R E L ATES OF EXPERIMENTAL BRAIN ISCHEMIA: QUANTIFIED EEG ANALY S I S Paola Bo, Debora Soragna, Claudia Specchia*, Pierluigi Chimento Laboratory of Experimental Neurophysiopathology and Neuropsychopharmacology, University of Pavia, IRCCS C. Mondino Institute of Neurology, Pavia; * Department of Health Sciences, Biostatistics Unit, University of Genoa, Italy Reprint requests to: Dr Paola Bo, Department of Neurological Sciences - University of Pavia, IRCCS C. Mondino Institute of Neurology, Via Palestro, 3-27100 Pavia, Italy. E mail: pbo@unipv.it Many environmental and occupational chemicals are known to affect the central and/or peripheral nervous system, causing changes that may result in neurological and psychiatric disorders. Because of the limited accessibility of the mammalian nervous tissue, new strategies are being developed to identify biochemical parameters of neuronal cell function, which can be measured in easily obtained tissues, such as blood cells, as potential markers of the chemically-induced alterations occurring in the nervous system. This review includes a comparative analysis of the effects of mercurials on calcium signalling in the neuroadrenergic PC12 cells and rat splenic T lymphocytes in an attempt to characterize this second messenger system as the sigma binding sites, as biological markers of psychiatric disorders is also discussed. KEY WORDS: Cerebral ischemia, quantified EEG analysis (QEEG), rabbit, rat. FUNCT NEUROL 01;16 (SUPPL.): 143-148 INTRODUCTION Cerebral ischemia of thrombotic or embolic origin is still a major cause of morbidity and m o r t a l i t y, mainly because the cellular mechanisms that lead to primary and secondary brain damage are still incompletely understood. This has stimulated many authors to develop experimental models to study the modification of biological and neurophysiological parameters in cerebral ischemia. The most common techniques are: microsurgical occlusion of the middle cerebral artery (MCA) by transorbital approach (1,2); intravascular embolization by microspheres or by homologous blood emboli (3-6); photothrombosis induced by Rose Be n g a l dye (7). THE INTRAVASCULAR EMBOLIZAT I O N METHOD In recent years, at the C. Mondino Institute of Neurology in Pavia, we have studied experimental cerebral ischemia in rabbits using the intravascular embolization method. The embolization was obtained by means of the infusion of specially prepared microspheres (.30-.35 mm in diameter, Eurand, Italy) through a catheter inserted in the ostium of the right internal carotid artery (Fig. 1, see over). The injection of 1-5-10 microspheres made it possible to obtain various degrees of cerebral ischemia (focal, multifocal, massive embolization). Looking at the time course of electroencephalogram (EEG), quantified EEG analysis (QEEG), somatosensory evoked potential (SEP), cerebral blood flow (CBF) and histological changes, we FUNCTIONAL NEUROLOGY (16) SUPPL. 4 01 143

P. Bo et al. found that the parameters examined were characterized by a peculiar pattern in each group. In part i c u l a r, the QEEG changes correlated well with the brain damage induced. In the group with focal ischemia, the increase in the slowest frequencies (delta activity) (Fig. 2) corresponded to a loss of cortical components of the SEPs (Fig. 3) and changes in CBF (Fig. 4). Similar changes, but more marked, were observed in the multifocal ischemia (Fig.s 5 and 6) and massive embolization (Fig.s 7 and 8) groups. The pathological pictures e m e rging in the 3 experimental groups ranged from a single ischemia, ipsilateral to the injected side, to widespread lesions and to massive brain edema (8) (Fig.s 9 and 10, over). The embolization method was found to be a good technique for developing different models of cerebral stroke (focal, multifocal ischemia and massive diffuse cerebral edema), as it resembles most closely the clinical situation in man. Using this experimental method we were able to evaluate changes in coagulability (9) and k opioid receptor binding (10) secondary to experimental cerebral ischemia in the rabbit. EEG and QEEG proved useful for analyzing and monitoring in vivo the evolution of cerebral i s c h e m i a. PHOTOTHROMBOSIS INDUCED BY ROSE BENGAL DYE More recently, we have considered the model of photochemically induced focal cerebral ischemia, in rats. This well established method is based on photochemical induction of thrombotic stroke, using Rose Bengal dye administered intravenously (7). It has the advantage of being less invasive and traumatic than the other methods. Owing to the complexity of the pathophysiological mechanisms involved in the evolution of cerebral ischemic events, several experimental studies, performed in different models, have been carried out in order to hypothesize new therapeutic strategies. As EEG monitoring has, in previous studies, shown a good correlation between the evolution of ischemic events and changes in cerebral electrical activity, both in the cerebral area submitted to stroke and in the contralateral F i g. 1 - Macroscopic appearance of embolization by microsphere in a branch of the right middle cerebral artery. RELATIVE 70% DENSITY 70% SPECTRUM µv 2 /HZ 12.. HZ 7. 12. HZ 3.70 7. HZ 0.15-3.70 HZ BASAL EM EMBOLISM F i g. 2 - QEEG in the group with focal ischemia. R i g h t : right cerebral hemisphere, Left: left cerebral hemisphere. R 10ms x P<0,05 p < 0.01 p < 0.001 Bas RIGHT Fig. 3 - SEPs of a rabbit of group 2 showing progressive loss of cortical components on the right hemisphere. LEFT 1h 4h 10 V 24h 1H 4H L 24H 144 FUNCTIONAL NEUROLOGY (16) SUPPL. 4 01

Experimental brain ischaemia: quantified EEG cpm x 10 2 30 10 5 A IV RELATIVE 12.. Hz 7. 12. Hz 3.70 7. Hz x P<0,05 p < 0.01 p < 0.001 2 1 10 5 2 III II I 1 5 10 15 min cpm x 10 2 B 70% 0.15-3.70 Hz 1 1 5 10 15 min F i g. 4 - A: Typical tracing of 133Xe wash-out curves according to ischemic lesion degree (groups I, II, III, IV) one hour after embolization.b: Typical tracing of 133Xe washout curves recorded four hours after embolization (groups II, III). II III DENSITY 70% SPECTRUM RIGHT BASAL EMBOLISM 1H Fig. 7 - QEEG in the group with massive embolization. Right: right hemisphere, Left: left hemisphere. LEFT RELATIVE 12.. Hz 7. 12. Hz x P<0,05 p < 0.01 p < 0.001 R L 3.70 7. Hz 70% 0.15-3.70 Hz Bas 10 V % DENSITY SPECTRUM RIGHT LEFT 10ms 1h BASAL EM EMEBOLISM Fig. 5 - QEEG in multifocal ischemia. Right: right cerebral hemisphere, left: Left cerebral hemisphere. 1H 4H Fig. 8 - SEPs of a rabbit with massive embolization. Right: right hemisphere, Left: left hemisphere. R L Bas 1h 10 V 10ms 4h Fig. 6 - SEPs of a rabbit with multifocal ischemia. Right: right hemisphere, Left: left hemisphere. Fig. 9 - Spongy alteration and nuclear pyknosis 8 hours after embolization. FUNCTIONAL NEUROLOGY (16) SUPPL. 4 01 145

P. Bo et al. Fig. 10 - Ischemic lesions of the right temporal area seen 24 h after embolization with microsphere. area, we performed an experimental protocol to evaluate this hypothetical correlation in photochemically-induced ischemia in rats. In this study, we used the well established Rose Bengal dye method to induce cerebral focal ischemia. The above method is based on the photochemical induction of thrombotic stroke using Rose Bengal dye administered intravenously in the tail vein. Rose Bengal is the most eff i c i e n t known photodynamic generator of singlet molecular oxygen which causes the formation of hydroperoxides in insaturated lipids (fatty acid autoxidation) (11). Photochemically induced cortical infarction has been demonstrated to be the consequence of primary endothelial membrane damage, which causes microvascular injury. The activation of platelet aggregation caused platelet thrombi within pial and parenchymal vessels, acutely depressing local cerebral blood flow (LCBF). The early blood-brain barrier breakdown increased brain water, which began at 15 min after irradiation, reached maximum values at 2 h and was still present at 5 days. The increase in [Na + ] and decrease in [K + ] was evident at 2 h, reached maximum values at 24 h, and was still present at 5 days (12). Another pathophysiological mechanism is glial swelling, which causes mechanical compression of adjacent microvessels. Similar results have been obtained by Lee et al. (13) using magnetic resonance imaging and examination of histological changes. Both techniques revealed reproducible primary and secondary damage characteristics. The time course of the changes characterizing the evolution of the ischemic event in the Rose Bengal experimental model of brain ischemia has been studied by several authors. Yi n et al. (14) investigated the role of GLAST mrna (a subtype of the glutamate transporter system), which plays an important role in some pathological conditions (15). These authors postulated that elevation of GLAST mrna expression in the cortex near the ischemic territory between 24 h and 72 h postischemia is correlated with the neuropathological process following the injury and may reflect a compensatory mechanism. Inhibition of protein synthesis in the ischemic area has long been demonstrated (16). In our study the EEG and the QEEG showed a significant increase in the slowest frequency bands (above all delta activity and to a lesser degree theta activity) in the cerebral area submitted to photochemical reaction and in the perilesional area. In the contralateral area, the increase in delta activity was evident 48-72 hours after the induction of cerebral ischemia (Fig.s 11-13). The values emerging from our statistical analysis are reported in Tables I-III. FrPaM left Fr right FrPaM right groud F i g. 11 - FrPaM right: area submitted to photochemical reaction. Fr right: perilesional area. FrPaM left: contralateral area. 146 FUNCTIONAL NEUROLOGY (16) SUPPL. 4 01

Experimental brain ischaemia: quantified EEG 1 100 80 60 40 0 basal 5 h 24 h 48 h 72 h Fig. 12 - QEEG in the area submitted to photochemical reaction (right Fr- PaM). 1 100 80 60 40 0 basal 5 h 24 h 48 h 72 h delta band band beta band theta alpha band F i g. 13 - QEEG in the perilesional area. 1 100 80 60 40 0 basal 5 h 24 h 48 h 72 h delta band band beta band theta alpha band F i g. 14 - QEEG in the contralateral area. Table I - Statistical significance of the changes in power density values obtained over time in the four band frequencies in the ischemic area. basal vs 5 h p<0.000 p<0.000 p<0.000 p<0.000 basal vs 24 h p<0.000 p<0.000 p<0.000 p<0.000 basal vs 48 h p<0.000 p<0.002 p<0.004 p<0.000 basal vs 72 h p<0.000 p<0.000 p<0.000 p<0.000 Table II - Statistical significance of the changes in power density values obtained over time in the four band frequencies in the perilesional ischemic area. basal vs 5 h p<0.01 n.s. p<0.001 p<0.000 basal vs 24 h p<0.01 n.s. n.s. n.s. basal vs 48 h p<0.000 p<0.001 n.s. n.s. basal vs 72 h p<0.001 p<0.001 p<0.001 p<0.01 Abbreviations: n.s. = not significant. Table III - Statistical significance of the changes in power density values obtained over time in the four band frequencies in the contralateral area. basal vs 5 h n.s. n.s. p<0.003 p<0.001 basal vs 24 h n.s. n.s. n.s. n.s. basal vs 48 h p<0.002 n.s. n.s. n.s. basal vs 72 h p<0.04 n.s. n.s. p<0.003 Abbreviations: n.s. = not significant. FUNCTIONAL NEUROLOGY (16) SUPPL. 4 01 147

P. Bo et al. CONCLUDING REMARKS We have considered two different methods in two different animal species. Although the mechanisms involved in the pathophysiology of the two models were different, the changes in cerebral electrical activity were similar. The time course of the evolution of ischemic events monitored by QEEG correlated well with biochemical and histological findings. ACKNOWLEDGMENTS This work was supported by the Grant ICS030.9/RC00 REFERENCES 11. Iannotti F, Hoff JT, Schielke GP. Brain tissue pressure in focal cerebral ischaemia. J Neurosurg 1985;62:83-89 12. Rosner G, Graf R, Kataoka K, Heiss WD. Selective functional vulnerability of cortical neurones following transient MCA-occlusion in the cat. Stroke 1986;17:76-81 13. Hegedus K, Fekete I, Tury F, Molnar L. Experimental focal cerebral ischaemia in rabbits. J Neurol 1985;232:223-230 14. Kaneko D, Nakamura N, Ogawa T. Cerebral infarction in rats using homologous blood emboli: development of a new experimental model. Stroke 1985;16:76-84 15. Kudo M, Aoyama A, Ichimori S, Fukanaga N. An animal model of cerebral infarction. Homologous blood clot emboli in rats. Stroke 1982;13:505-508 16. Widing O. Cerebral microembolization following carotid injection of dextran microspheres in rabbits. Neuroradiology 1981;21: 1 2 3-1 2 6 17. Watson BD, Dietrich WD, Busto R, Wa c h t e l MS, Ginsberg MD. Induction of reproducible brain infarction by photochemically initiated thrombosis. Ann Neurol 1985;17:497-504 18. Bo P, Cosi V, Introzzi G et al. Quantified EEG, somatosensory evoked potentials and cerebral blood flow in monitoring experimental brain ischaemia. Ital J Neurol Sci 1 9 8 7 ; 8 : 5 4 9-5 5 9 19. Cipolli PL, Bo P, Marchioni E, Matti C, Savoldi F, Taccola A. Blood hypercoagulability secondary to experimental cerebral ichemia in the rabbit: influence of a hyperdyslipemia induced by an atherogenic diet. Ital J Neurol Sci 1991;12:289-293 10. Scavini C, Rozza A, Bo P et al. κ-opioid receptor changes and neurophysiological alterations during cerebral ischaemia. Stroke 1 9 9 0 ; 2 1 : 9 4 3-9 4 7 11. Pooler JP, Valenzeno DP. Dye-sensitized photodynamic inactivation of cells. Med Phys 1 9 8 1 ; 8 : 6 1 4-6 2 8 12. Dietrich WD, Busto R, Watson BD, Scheinb e rg P, Ginsberg MD. Photochemically induced cerebral infarction. II. Edema and blood-brain barrier disruption. Acta Neuropathol (Berl) 1987;72:326-334 13. Lee VM, Burdett NG, Carpenter A et al. Evolution of photochemically induced focal cerebral ischaemia in the rat. Magnetic resonance imaging and histology. Stroke 1 9 9 6 ; 2 7 : 2 11 0-2 11 8 14. Yin KJ, Yan YP, Sun FY. Altered expression of glutamate transporter GLAST mrna in rat brain after photochemically induced focal ischaemia. Anat Rec 1998;251:9-14 15. Nicholls D, Attwell D. The release and uptake of excitatory amino acids. Tr e n d s Pharmacol Sci 1990;11:462-468 16. Hossmann KA. Disturbances of cerebral protein synthesis and ischaemic cell death. Prog Brain Res 1993;96:161-177 148 FUNCTIONAL NEUROLOGY (16) SUPPL. 4 01