Pharmacoeiectroencephalography
Original Paper Neuropsychobiology 1992:26:108-112
Department of Pharmacology and Toxicology. Faculty of Pharmacy. Higher Medical Institute, Sofia. Bulgaria
KeyW ords Somatosensory' evoked potential Endothelin Cerebral ischemia
Effect of Endothelin on Somatosensory Evoked Potentials in Rats
Abstract
The effects of endothelin 1 (ET-1; 300pmol/rat intracarotid) on somatosen sory evoked potential were investigated in rats. ET-1 led to an amplitude reduction, peak latency prolongations and waveform disturbances. There was a large interindividual variability. The late cortical components were more affected than the earlier potentials at a thalamic or cortical level. ET-1induccd SEP changes developed quickly after the drug injection and persisted for at least 30 min. It is assumed that the observed effects probably reflect the occurrence of a progressively developing ischemia subsequent to ET-1 admin istration. Moreover, the pattern of ET-1-induced changes suggests a greater sensitivity of the synaptic transmission to the ischemic influence than the axonal conduction.
Introduction
After the discovery of endothelin 1 (ET-1), one of the most potent vasoconstrictive peptides, produced by the vascular endothelial cells [ 1 ]. a large number of investiga tions were performed in order to reveal its main biological properties. It has been established recently that ET-1 elic its slow developing and extremely long-lasting vasocon striction of cerebral and peripheral arteries in vitro and in vivo [1-11]. Consequently some authors have shown that ET-1 is able to induce a reduction of the cerebral perfu sion [5, 12], It is found that the reduction of the local cere bral blood flow in experimental conditions results in a correlative suppression of somatosensory evoked poten tials [13, 14], Since the information on the electrophysiological changes, especially of evoked potentials, following ET-1
administration, is insufficient, our study was designed to determine the ET-1 effect on somatosensory evoked po tential (SEP) in rats.
Material and Methods Seven mature male rats (Wistar) weighing 291 ± 10 g were used. The investigations were carried out in acute experiments. All opera tions were done under ether anesthesia. Animals were tracheotomized and artificially respirated with room air at a rate of 35 strokes/ min and 5 ml/stroke. Complete relaxation was obtained by repeated intravenous ¡-/-tubocurarin injections via the cannulated v. dorsalis penis. The animals were then placed atraumatically in a stereotactic frame. After skull exposure, small (< 1 mm) monopolar electrodes (Ag/ AgCI balls) were fixed in an epidural position with the recording elec trode placed over the left somatosensory area of the cerebral cortex (3-4 mm lateral and I mm anterior to the bregma - just rostral to the
Dr. Anclia Todorova Department o f Pharmacology and Toxicology Faculty of Pharmacy. Higher Medical Institute 2. Dunav Street
© 1992 S. Karger AG. Basel 0302-282X/92/0262-0I08 S2.75/0
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A. Todorova M. Boyagieva T. Yosifov
were drowsy during the short (150 min) recording period following the discontinuation of the anesthetic. It is suggested that the animals were not in great discomfort but further investigations should proba bly require a more complete anesthetic procedure.
Results
In table 1 the mean autocontrol values of the main parameters (peak latency and peak-to-peak amplitude) of P40N90 complex and the respective values at the 5th min after ET-1 administration in the 7 tested rats are present ed. Three of the examined animals (rats 1. 2 and 3) showed significant morphological changes in all SEP components. This started from a strong amplitude de crease and a peak latency increase to non-determinable peaks and a lack of intermediate and late components (rat 1) already in the first (1-5) min subsequent to ET-1 administration (fig. 1A). The early III and P|. were pre served. although with changed parameters. In one of these rats (rat 3) the latency alterations in the early and interme diate potentials developed a little later in the time-course (from the 20th min). The SEP changes, induced by ET-1, persisted to the end of the experiments (90 min after ET-1 administration). In 1 animal (rat 4) only the intermediate and late cortical components changed (exceeding 2 SD of the mean autocontrol value): there was a decrease of PvNv amplitude from the 5th to the 90th min. a discreet reduc tion ofNixPju amplitude (from the 30th min) and a prom inent widening and flattening of P40N90 complex with a complete absence ofN 9o at the 1st min (fig. IB). The peak latencies of P40 and N90 increased from the 1st to the 5th min to the end of the observation period (90 min after ET-1 injection). P40N90 was strongly suppressed at the 1st min with a transient recovery of the initial state at the 5th min and a subsequent prolonged amplitude decrease (from the 20th to the 90th min). Two of the examined animals (rats 5 and 6) showed more discreet changes. They started with a transient (from the 1st to the 5th min) latency increase in the early (III and P() and intermediate components, followed in rat 6 by a slight latency shortening in the subsequent minutes (to the 90th min). At the same time the late potentials reacted with a prolonged latency increase: there was an elongation of P40 and N90 latency from the 1st to the 30th min in rat 6, a latency increase of N !8 and P4() from the 20th min and of N90 from the 45th to the 90th min in rat 5 (fig. 1C). ET-1 injection resulted also in a prominent amplitude reduction of P40N90complex at the 1st min (rat 5) or from the 1st to the 90th min (rat 6). These changes were accompanied by an amplitude reduction of the early
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coronal suture). This location corresponds to position 5 used by Wicderholt and Iragui-Madoz [15] and is the same as that used by Shaw and Cant [16]. The reference electrode was inserted into the nasal bone. The left external carotid artery was cannulatcd retrogradcly for intracarotid (i.c.) drug injection [12], All wound margins and pres sure points were repeatedly infiltrated with 0.5% procain and the rectal temperature was maintained at about 37 °C. The anaesthetic was discontinued and the animals were allowed to recover from the surgical procedures for the next 30-40 min. The contralateral fore paw was stimulated using 2 hypodermic stainless steel needle elec trodes inserted beneath the volar surface of its distal part. Isolated constant current square waves of 0.05-ms duration were used. The intensity of the electrical stimulation was sufficient to produce a clearly defined paw twitch in the same animal before curarization. SEPs were recorded with a polyphysiograph and were fed through an ADC (analog-to-digital converter) into a computer for further data processing with an appropriate program [17], The sweep durations were 25.6 ms (0.2-ms intersample interval, 5.3-3.000 Hz bandpass setting. 3-Hz stimulus rate, 100 responses averaged) and 256 ms (1ms intersamplc interval. 5.3-35 Hz bandpass setting, 0.3-Hz stimu lus rate. 20 responses averaged). SEPs were registered at the two dif ferent sweep durations in an immediate sequence at every observed minute in order to obtain a record of all main components (early and late) in one and the same animal at every moment of a given experi ment. Changes in the waveform, peak latencies and amplitudes of the SEP components were recorded. At the sweep duration of 25.6 ms the following SEP waves were considered: III. Pi, N|. P; and [15, 16]. With the sweep duration of 256 ms the observed negativc-positive-negative sequence was assigned as N,«. P40 and N90 on the basis of the calculated mean latency values of these components in the 7 tested animals. The components of the evoked response have been classified as ‘early’ (III. P t and N|). ‘intermediate’ (Pi and Ni) and ‘late potentials'(Nig. P ^ a n d Nw) for the purpose of description. The amplitudes were measured peak-to-baseline for peaks III and P|. and as peak-to-peak amplitudes for the rest (P|N t, P:Ni, NigP.m and P40N90). Using the described electrophysiological method SEP parameters were evaluated after administration of ET-I (300pmol/ral i.c.) in accordance w ith the study of Willette et al. [12], ET-1 was prepared in saline and administered i.c. in volumes of 0.15 ml. The druginduced SEPs were recorded I, 5. 20. 30. 45. 60 and 90 min after ET-1 injection. Every animal served as its own control (autocontrol). Three averaged SEPs were recorded during the first 30 min of each experiment (following the adaptation period and before the peptide injection). The mean values of the SEP parameters from these three records served as a base for further comparison of the subsequent alterations in the evoked response. Only these SEP parameters, the values of which subsequent to ET-1 administration exceeded 2 stan dard deviations (2 SD) of the mean autocontrol value were consid ered changed by the peptide. Afterwards, the obtained data under went a statistical analysis with the 2-tailed Wilcoxon matched-pairs test [18]. ET-1 (human/porcine) was obtained from the Peptide Insti tute Inc.. Osaka. Japan. In performing the present study it was necessary to immobilize the animals with a minimal influence of the anesthetic on the evoked response. On the basis of the well known anesthetic properties of ether, providing a prolonged recovery of consciousness from the anesthesia [19], it could be assumed that the investigated animals
Fig. 1. Effects of ET-I on SEP at a sweep duration of 256 ms (n = I, 20 responses averaged) in 3 exemplary rats (A. B and C). The thick line represents the control potentials and the dotted line represents SEPs 60 min (A). I min (B) and 30 min (C) after ET-l administration (300 pmol/rat i.c.).
Table 1. Original values of the peak latencies and peak-to-peak amplitude of P40N90 com plex in the control period (means ± 2 S D o fth e 3 predrug SEP records during the first 30 min in each experiment) and the respective values at the 5th min after ET-l administration (300 pmol/rat i.c.) in the 7 tested rats
Rat No.
Time
Latency, ms
I
Control ± 2 SD 5 min ET-l
50.33 ±3.06
95.33 ±19.00
124.03 ±22.88
2
Control ± 2 SD 5 min ET-l
55.33 ±3.06 78.13
104.00 ±10.00 131.00
116.37 ±55.10 4.93
3
Control ± 2 SD 5 min ET-l
30.67 ±1.16 58.00
74.67 ±2.30 88.00
60.70 ±20.10 25.67
4
Control ± 2 SD 5 min ET-l
57.67 ±8.32 94.00
133.67± 13.32 220.00
116.35 ± 51.62 117.27
5
Control ± 2 SD 5 min ET-l
40.50±4.24 40.00
74.50+21.22 77.00
152.58 ±9.24 106.52
6
Control ± 2 SD 5 min ET-l
42.33 ±3.06 52.00
84.00 ±2.00 97.00
168.45 ±56.58 124.79
7
Control ± 2 SD 5 min ET-l
44.67 ±6.16 42.00
98.33 ± 14.18 103.00
169.50 ±52.42 185.10
Amplitude, )tV N»
P40
P40N90
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T odorova/Boyagi eva/Y osi fov
SEP and Endothelin
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The dash indicates that the corresponding peak is absent in the SEP waveform.
Discussion
The performed experiments showed that ET-1 (300pmol/rat i.c.) led to certain SEP changes, generally expressed by latency prolongations and amplitude reduc tions to lack of the evoked response. ET-1-induced SEP changes predominantly concerned the late cortical com ponents and were characterized by a large interindividual variability in their degree of expression. SEP alterations, induced by ET-1. could be assigned to different times fol lowing the peptide injection, but more often they were observed in the first minutes and lasted till the end of the experiments (or for at least 30 min). The different reactions to ET-1 effect on SEP might be related to the grades of the cortical SEP abnormalities scale provided by Ahmed [20] for comatose patients. ET-1 elicited strong changes in the cortical SEPs. It is accepted that peak III is generated in or near the thalamus and the thalamocortical sensor radiation and P| repre sents the primary response of the somatosensory receiv ing area [15. 16. 21 ]. The early SEP components (III. Pi and N|) were less or not affected by ET-1. Consequently the late cortical components are more sensitive to ET-1 influence whereas the short-latency potentials at the tha lamic and cortical level are more stable. According to Willette et al. [ 12] ET-1 administration in a dosage of 300 pmol/rat i.c. leads to a reduction of the cerebral perfusion up to 40% from the baseline. There are a large number of investigations concerning SEP changes during ischemia. It is demonstrated that there is a de crease in the amplitudes and an increase in the peak laten cies of the cortical SEP components, while the preceding waves reflecting the subcortical activity are normal [13, 14, 20, 22-28]. It is also found that there is a close rela
tionship between the reduction of the cerebral blood flow and the respective SEP suppression in experimental con ditions [13, 14], On the basis of the foregoing, given the potent vasoconstrictive properties of ET-1, we suggest that the SEP changes observed in our study are the result of a progressively developed ischemia produced by this peptide. ET-1 fails to enter the brain tissue following a systemic administration [29], Consequently ET-1 effects on SEP most probably reflect the reduced cerebral perfu sion and the developed brain ischemia and are not a result of direct neuronal effects in the central nervous system (CNS). ET-1-induced SEP changes developed quickly af ter injection and an entire recovery of the initial state was not achieved for the whole study period. Chabrier et al. [30] found that ET-1 binds with high affinity receptors in the vascular smooth muscle cells and its binding is very tight and almost impossible to dissociate. It is possible that the stable binding of ET-1 to the respective vascular receptors underlies the prolonged vasoconstrictive effect of this peptide. It is assumed that the large interindividual variability of ET-1 effects on SEP observed in our study could be assigned to some differences in the number and affinity of the vascular ET-1 binding sites in the tested animals, which determine their different sensitivity and reactivity to ET-1 influence. The possible explanation of the differences in sensitivity of the late and early SEP components to ET-1 influence could be that the synaptic transmission is affected by the induced ischemia to a greater extent than the axonal conduction. Probably be cause of that, the polysynaptic late cortical components were more prominently altered by ET-1. Our results suggest that SEP is very sensitive to ET-1 effects on CNS and the applied method could serve as a tool for further, more detailed studies on cerebral isch emia.
Acknowledgements The authors wish to express their gratitude to the Peptide Insti tute Inc.. Osaka. Japan, for the generous supply of endothelin used in this study.
I ll
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and intermediate components (especially III and P| in both rats from the 1st to the 30th min with a subsequent transient increase of the amplitudes in rat 6. In 1 animal (rat 7) there were no substantial changes in SEP following ET-1 administration, except for a latency shortening in the intermediate potentials and the late N |g from the 20th to the 60th min. Applying the 2-tailed Wilcoxon matched-pairs test, we found a statistically significant amplitude reduction of P40N90 from the 1st to the 90th min after ET-1 injection (p < 0.05) and a significant prolongation of N90 latency at the 5th, 20th and 60th min after ET-1 (p < 0.05), for the parameters of P40N90 complex changed unidirectionally in 6 of the 7 tested animals.
1 Yanagisawa M. Kurihara H. Kimura S, Tomobe Y. Kobayashi M. Mitsui Y. Yazaki Y. Goto K. Masaki T: A novel potent vasocon strictor peptide produced by vascular endothe lial cells. Nature 1988:332:411-415. 2 Mima T. Yanagisawa M. Shigeno T. Saito A. Goto K. Takakura K. Masaki T: Endothelin acts in feline and canine cerebral arteries from the adventitial side. Stroke 1989:20:1553— 1556. 3 Kadel KA. Heistad DD, Faraci FM: Effects of endothelin on blood vessels of the brain and choroid plexus. Brain Res 1990:518:78—82. 4 Robinson MJ. McCulloch J: Contractile re sponses to endothelin in feline cortical vessels in situ. J Cercb Blood Flow Mctab 1990:10: 285-289. 5 Robinson MJ. Macrae IM. Todd M. Reid JL. McCulloch J: Reduction of local cerebral blood flow to pathological levels by endothelin-1 ap plied to the middle cerebral artery in the rat. Neurosci Lett 1990:118:269-272. 6 Saito A. Mima T. Shigeno T. Yanagisawa M. Goto K, Kimura S. Masaki T: Endothelininduced vasoconstriction of cerebral and pe ripheral arteries of dogs. Eur J Pharmacol 1990:183:1803-1804. 7 Shirahase H. Usui H. Shimaji H. Kurahashi K. Fujiwara M: Endothelium-dependent and -independent contraction induced by endothclin-l in canine basilar arteries. EurJ Pharmacol 1990:183:1804-1805. 8 Tomobe Y. Ishikawa T. Yanagisawa M. Ki mura S. Masaki T. Goto K: Mechanisms of increased sensitivity to endothelin-1 aorta from spontaneously hypertensive rats. Eur J Pharmacol 1990:183:1817. 9 Willette RN. Sauermelch CF: Abluminal ef fects ofendothelin in cerebral microvasculature assessed by laser-Doppler flowmetry. Am J Physiol 1990:259:H 1688-H1693. 10 Vila JM. de Aguilera EM. Martinez MC. Ro driguez MD. Irurzun A, Lluch S: Endothelin action on goat cerebral arteries. J Pharm Phar macol 1990:42:370-372.
11 Saito A. Shiba R. Yanagisawa M. Masaki T. Kimura S. Yamada K. Mima T, Shigeno T. Goto K: Endothclins: Vasoconstrictor effects and localization in canine cerebral arteries. Br J Pharmacol 1991; 103:112 9 -1135. 12 Willclte RN. Sauermelch C, Ezekiel W. Feuerstcin G. Ohlstein EH: Effect of endothelin on cortical microvascular perfusion in rats. Stroke 1990:21:451-458. 13 Branston NM. Symon L. Crockard HA, Pasztor E: Relationship between the cortical evoked potential and local cortical blood flow' follow ing acute middle cerebral artery occlusion in the baboon. Exp Neurol 1974:45:195-208. 14 Kochs E. Schulte am Esch J: Alterations in somatosensory' evoked responses following progressive incomplete ischemia in goats (ab stracts A0637). 9th World Congr Anesthesiol. Washington. May 22-28, 1988. 15 Wiedcrholt WC. Iragui-Madoz VJ: Far field somatosensory potentials in the rat. Elcctroenceph Clin Neurophvsiol 1977:42:456—465. 16 Shaw N A. Cant BR: The effect of pentobarbital on central somatosensory conduction time in the rat. Elcctroenceph Clin Neurophysiol 1981: 51:674-677. 17 Daskalova Ml: Wave analysis of spontaneous and evoked neurophysiological signals using microcomputer systems: doctoral diss. Bulgar ian Academy of Sciences. Sofia, 1988. 18 l.entncr C (ed): Introduction to Statistics. Sta tistical Tables. Mathematical Formulae. Geigy Scientific Tables, ed 8. Basle. Ciba-Geigy. 1982, vol 2. 19 Rang H. Dale M: Pharmacology, ed 2. Edin burgh. Churchill Livingstone. 1991. 20 Ahmed I: Use of somatosensory evoked re sponses in the prediction of outcome from coma. Clin Electroenceph 1988:19:78-86. 21 Budnick B. McKeown KL. Wiederholt WC: Hypothermia-induced changes in rat short la tency somatosensory' evoked potentials. Elec troenceph Clin Neurophvsiol 1981:51:19-31.
22 Anziska BJ. Cracco RQ: Short latency somato sensory' evoked potentials in brain dead pa tients. Arch Neurol 1980:37:222-225. 23 Jones SJ: Clinical applications of short-latcncv somatosensory' evoked potentials. Ann NY AcadSci 1982:388:369-387. 24 Walser H, Mattie H. Keller HM. Janzer R: Early cortical median nerve somatosensory evoked potentials. Prognostic value in anoxic coma. Arch Neurol 1985:42:32-38. 25 Amantini A. de Scisciolo G. Bartcli M. Lori S. Ronchi O, Prates C. Pinto F: SF.P changes related to cerebral ischemia during carotid sur gery'. Clin Neurol Neurosurg I987:89(suppl l):30. 26 Gilbert J. Firsching R. Bunegin L. Gelineau J: The relative value of different neurophysiologi cal monitors in cerebral ischemia (abstract A0632). 9th World Congr Anesthesiol. Wash ington. May 22-28. 1988. 27 Russ W. Thiel A. Moosdorf R. Hcmpclmann G: Somatosensorisch evoziertc Potentiale bei descblitcriercnden Eingriffcn an der Carotisgabel. Klin Wochenschr I988:66(suppl I4):3540. 78 Rothstein TL. Thomas EM. Sumi SM: Predict ing outcome in hvpoxic-ischcmic coma. A pro spective clinical and electrophysiologic study. Electroenceph Clin Neurophysiol 1991:79: 101-107. 29 Stasch JP. Steinke W. Kazda S. Meuser D: Autoradiographic localization of i:5l-endothelin in rat tissues. Arzncimittelforschung 1989: 39:59-61. 30 Chabrier P-E, Lonchampt M-O, Roubert P. Pinelis V. Auguct M. Guillon JM, Viossat I. Pirotzky E. Hosford D. Braquet P: Intracellular signalling pathways and binding of endothelin in vascular smooth muscle cells. EurJ Pharma col 1990:183:676.
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References
Original Paper Neuropsychobiology 1992:26:108-112
Department of Pharmacology and Toxicology. Faculty of Pharmacy. Higher Medical Institute, Sofia. Bulgaria
KeyW ords Somatosensory' evoked potential Endothelin Cerebral ischemia
Effect of Endothelin on Somatosensory Evoked Potentials in Rats
Abstract
The effects of endothelin 1 (ET-1; 300pmol/rat intracarotid) on somatosen sory evoked potential were investigated in rats. ET-1 led to an amplitude reduction, peak latency prolongations and waveform disturbances. There was a large interindividual variability. The late cortical components were more affected than the earlier potentials at a thalamic or cortical level. ET-1induccd SEP changes developed quickly after the drug injection and persisted for at least 30 min. It is assumed that the observed effects probably reflect the occurrence of a progressively developing ischemia subsequent to ET-1 admin istration. Moreover, the pattern of ET-1-induced changes suggests a greater sensitivity of the synaptic transmission to the ischemic influence than the axonal conduction.
Introduction
After the discovery of endothelin 1 (ET-1), one of the most potent vasoconstrictive peptides, produced by the vascular endothelial cells [ 1 ]. a large number of investiga tions were performed in order to reveal its main biological properties. It has been established recently that ET-1 elic its slow developing and extremely long-lasting vasocon striction of cerebral and peripheral arteries in vitro and in vivo [1-11]. Consequently some authors have shown that ET-1 is able to induce a reduction of the cerebral perfu sion [5, 12], It is found that the reduction of the local cere bral blood flow in experimental conditions results in a correlative suppression of somatosensory evoked poten tials [13, 14], Since the information on the electrophysiological changes, especially of evoked potentials, following ET-1
administration, is insufficient, our study was designed to determine the ET-1 effect on somatosensory evoked po tential (SEP) in rats.
Material and Methods Seven mature male rats (Wistar) weighing 291 ± 10 g were used. The investigations were carried out in acute experiments. All opera tions were done under ether anesthesia. Animals were tracheotomized and artificially respirated with room air at a rate of 35 strokes/ min and 5 ml/stroke. Complete relaxation was obtained by repeated intravenous ¡-/-tubocurarin injections via the cannulated v. dorsalis penis. The animals were then placed atraumatically in a stereotactic frame. After skull exposure, small (< 1 mm) monopolar electrodes (Ag/ AgCI balls) were fixed in an epidural position with the recording elec trode placed over the left somatosensory area of the cerebral cortex (3-4 mm lateral and I mm anterior to the bregma - just rostral to the
Dr. Anclia Todorova Department o f Pharmacology and Toxicology Faculty of Pharmacy. Higher Medical Institute 2. Dunav Street
© 1992 S. Karger AG. Basel 0302-282X/92/0262-0I08 S2.75/0
Downloaded by: King's College London 137.73.144.138 - 1/24/2019 6:48:38 PM
A. Todorova M. Boyagieva T. Yosifov
were drowsy during the short (150 min) recording period following the discontinuation of the anesthetic. It is suggested that the animals were not in great discomfort but further investigations should proba bly require a more complete anesthetic procedure.
Results
In table 1 the mean autocontrol values of the main parameters (peak latency and peak-to-peak amplitude) of P40N90 complex and the respective values at the 5th min after ET-1 administration in the 7 tested rats are present ed. Three of the examined animals (rats 1. 2 and 3) showed significant morphological changes in all SEP components. This started from a strong amplitude de crease and a peak latency increase to non-determinable peaks and a lack of intermediate and late components (rat 1) already in the first (1-5) min subsequent to ET-1 administration (fig. 1A). The early III and P|. were pre served. although with changed parameters. In one of these rats (rat 3) the latency alterations in the early and interme diate potentials developed a little later in the time-course (from the 20th min). The SEP changes, induced by ET-1, persisted to the end of the experiments (90 min after ET-1 administration). In 1 animal (rat 4) only the intermediate and late cortical components changed (exceeding 2 SD of the mean autocontrol value): there was a decrease of PvNv amplitude from the 5th to the 90th min. a discreet reduc tion ofNixPju amplitude (from the 30th min) and a prom inent widening and flattening of P40N90 complex with a complete absence ofN 9o at the 1st min (fig. IB). The peak latencies of P40 and N90 increased from the 1st to the 5th min to the end of the observation period (90 min after ET-1 injection). P40N90 was strongly suppressed at the 1st min with a transient recovery of the initial state at the 5th min and a subsequent prolonged amplitude decrease (from the 20th to the 90th min). Two of the examined animals (rats 5 and 6) showed more discreet changes. They started with a transient (from the 1st to the 5th min) latency increase in the early (III and P() and intermediate components, followed in rat 6 by a slight latency shortening in the subsequent minutes (to the 90th min). At the same time the late potentials reacted with a prolonged latency increase: there was an elongation of P40 and N90 latency from the 1st to the 30th min in rat 6, a latency increase of N !8 and P4() from the 20th min and of N90 from the 45th to the 90th min in rat 5 (fig. 1C). ET-1 injection resulted also in a prominent amplitude reduction of P40N90complex at the 1st min (rat 5) or from the 1st to the 90th min (rat 6). These changes were accompanied by an amplitude reduction of the early
109
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coronal suture). This location corresponds to position 5 used by Wicderholt and Iragui-Madoz [15] and is the same as that used by Shaw and Cant [16]. The reference electrode was inserted into the nasal bone. The left external carotid artery was cannulatcd retrogradcly for intracarotid (i.c.) drug injection [12], All wound margins and pres sure points were repeatedly infiltrated with 0.5% procain and the rectal temperature was maintained at about 37 °C. The anaesthetic was discontinued and the animals were allowed to recover from the surgical procedures for the next 30-40 min. The contralateral fore paw was stimulated using 2 hypodermic stainless steel needle elec trodes inserted beneath the volar surface of its distal part. Isolated constant current square waves of 0.05-ms duration were used. The intensity of the electrical stimulation was sufficient to produce a clearly defined paw twitch in the same animal before curarization. SEPs were recorded with a polyphysiograph and were fed through an ADC (analog-to-digital converter) into a computer for further data processing with an appropriate program [17], The sweep durations were 25.6 ms (0.2-ms intersample interval, 5.3-3.000 Hz bandpass setting. 3-Hz stimulus rate, 100 responses averaged) and 256 ms (1ms intersamplc interval. 5.3-35 Hz bandpass setting, 0.3-Hz stimu lus rate. 20 responses averaged). SEPs were registered at the two dif ferent sweep durations in an immediate sequence at every observed minute in order to obtain a record of all main components (early and late) in one and the same animal at every moment of a given experi ment. Changes in the waveform, peak latencies and amplitudes of the SEP components were recorded. At the sweep duration of 25.6 ms the following SEP waves were considered: III. Pi, N|. P; and [15, 16]. With the sweep duration of 256 ms the observed negativc-positive-negative sequence was assigned as N,«. P40 and N90 on the basis of the calculated mean latency values of these components in the 7 tested animals. The components of the evoked response have been classified as ‘early’ (III. P t and N|). ‘intermediate’ (Pi and Ni) and ‘late potentials'(Nig. P ^ a n d Nw) for the purpose of description. The amplitudes were measured peak-to-baseline for peaks III and P|. and as peak-to-peak amplitudes for the rest (P|N t, P:Ni, NigP.m and P40N90). Using the described electrophysiological method SEP parameters were evaluated after administration of ET-I (300pmol/ral i.c.) in accordance w ith the study of Willette et al. [12], ET-1 was prepared in saline and administered i.c. in volumes of 0.15 ml. The druginduced SEPs were recorded I, 5. 20. 30. 45. 60 and 90 min after ET-1 injection. Every animal served as its own control (autocontrol). Three averaged SEPs were recorded during the first 30 min of each experiment (following the adaptation period and before the peptide injection). The mean values of the SEP parameters from these three records served as a base for further comparison of the subsequent alterations in the evoked response. Only these SEP parameters, the values of which subsequent to ET-1 administration exceeded 2 stan dard deviations (2 SD) of the mean autocontrol value were consid ered changed by the peptide. Afterwards, the obtained data under went a statistical analysis with the 2-tailed Wilcoxon matched-pairs test [18]. ET-1 (human/porcine) was obtained from the Peptide Insti tute Inc.. Osaka. Japan. In performing the present study it was necessary to immobilize the animals with a minimal influence of the anesthetic on the evoked response. On the basis of the well known anesthetic properties of ether, providing a prolonged recovery of consciousness from the anesthesia [19], it could be assumed that the investigated animals
Fig. 1. Effects of ET-I on SEP at a sweep duration of 256 ms (n = I, 20 responses averaged) in 3 exemplary rats (A. B and C). The thick line represents the control potentials and the dotted line represents SEPs 60 min (A). I min (B) and 30 min (C) after ET-l administration (300 pmol/rat i.c.).
Table 1. Original values of the peak latencies and peak-to-peak amplitude of P40N90 com plex in the control period (means ± 2 S D o fth e 3 predrug SEP records during the first 30 min in each experiment) and the respective values at the 5th min after ET-l administration (300 pmol/rat i.c.) in the 7 tested rats
Rat No.
Time
Latency, ms
I
Control ± 2 SD 5 min ET-l
50.33 ±3.06
95.33 ±19.00
124.03 ±22.88
2
Control ± 2 SD 5 min ET-l
55.33 ±3.06 78.13
104.00 ±10.00 131.00
116.37 ±55.10 4.93
3
Control ± 2 SD 5 min ET-l
30.67 ±1.16 58.00
74.67 ±2.30 88.00
60.70 ±20.10 25.67
4
Control ± 2 SD 5 min ET-l
57.67 ±8.32 94.00
133.67± 13.32 220.00
116.35 ± 51.62 117.27
5
Control ± 2 SD 5 min ET-l
40.50±4.24 40.00
74.50+21.22 77.00
152.58 ±9.24 106.52
6
Control ± 2 SD 5 min ET-l
42.33 ±3.06 52.00
84.00 ±2.00 97.00
168.45 ±56.58 124.79
7
Control ± 2 SD 5 min ET-l
44.67 ±6.16 42.00
98.33 ± 14.18 103.00
169.50 ±52.42 185.10
Amplitude, )tV N»
P40
P40N90
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SEP and Endothelin
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The dash indicates that the corresponding peak is absent in the SEP waveform.
Discussion
The performed experiments showed that ET-1 (300pmol/rat i.c.) led to certain SEP changes, generally expressed by latency prolongations and amplitude reduc tions to lack of the evoked response. ET-1-induced SEP changes predominantly concerned the late cortical com ponents and were characterized by a large interindividual variability in their degree of expression. SEP alterations, induced by ET-1. could be assigned to different times fol lowing the peptide injection, but more often they were observed in the first minutes and lasted till the end of the experiments (or for at least 30 min). The different reactions to ET-1 effect on SEP might be related to the grades of the cortical SEP abnormalities scale provided by Ahmed [20] for comatose patients. ET-1 elicited strong changes in the cortical SEPs. It is accepted that peak III is generated in or near the thalamus and the thalamocortical sensor radiation and P| repre sents the primary response of the somatosensory receiv ing area [15. 16. 21 ]. The early SEP components (III. Pi and N|) were less or not affected by ET-1. Consequently the late cortical components are more sensitive to ET-1 influence whereas the short-latency potentials at the tha lamic and cortical level are more stable. According to Willette et al. [ 12] ET-1 administration in a dosage of 300 pmol/rat i.c. leads to a reduction of the cerebral perfusion up to 40% from the baseline. There are a large number of investigations concerning SEP changes during ischemia. It is demonstrated that there is a de crease in the amplitudes and an increase in the peak laten cies of the cortical SEP components, while the preceding waves reflecting the subcortical activity are normal [13, 14, 20, 22-28]. It is also found that there is a close rela
tionship between the reduction of the cerebral blood flow and the respective SEP suppression in experimental con ditions [13, 14], On the basis of the foregoing, given the potent vasoconstrictive properties of ET-1, we suggest that the SEP changes observed in our study are the result of a progressively developed ischemia produced by this peptide. ET-1 fails to enter the brain tissue following a systemic administration [29], Consequently ET-1 effects on SEP most probably reflect the reduced cerebral perfu sion and the developed brain ischemia and are not a result of direct neuronal effects in the central nervous system (CNS). ET-1-induced SEP changes developed quickly af ter injection and an entire recovery of the initial state was not achieved for the whole study period. Chabrier et al. [30] found that ET-1 binds with high affinity receptors in the vascular smooth muscle cells and its binding is very tight and almost impossible to dissociate. It is possible that the stable binding of ET-1 to the respective vascular receptors underlies the prolonged vasoconstrictive effect of this peptide. It is assumed that the large interindividual variability of ET-1 effects on SEP observed in our study could be assigned to some differences in the number and affinity of the vascular ET-1 binding sites in the tested animals, which determine their different sensitivity and reactivity to ET-1 influence. The possible explanation of the differences in sensitivity of the late and early SEP components to ET-1 influence could be that the synaptic transmission is affected by the induced ischemia to a greater extent than the axonal conduction. Probably be cause of that, the polysynaptic late cortical components were more prominently altered by ET-1. Our results suggest that SEP is very sensitive to ET-1 effects on CNS and the applied method could serve as a tool for further, more detailed studies on cerebral isch emia.
Acknowledgements The authors wish to express their gratitude to the Peptide Insti tute Inc.. Osaka. Japan, for the generous supply of endothelin used in this study.
I ll
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and intermediate components (especially III and P| in both rats from the 1st to the 30th min with a subsequent transient increase of the amplitudes in rat 6. In 1 animal (rat 7) there were no substantial changes in SEP following ET-1 administration, except for a latency shortening in the intermediate potentials and the late N |g from the 20th to the 60th min. Applying the 2-tailed Wilcoxon matched-pairs test, we found a statistically significant amplitude reduction of P40N90 from the 1st to the 90th min after ET-1 injection (p < 0.05) and a significant prolongation of N90 latency at the 5th, 20th and 60th min after ET-1 (p < 0.05), for the parameters of P40N90 complex changed unidirectionally in 6 of the 7 tested animals.
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