The Effect of Diazepam on Neonatal Seizure: in Vivo 31Pand 'H NMR Study1
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003 1-3998/89/2501-0027$02.00/0 PEDIATRIC RESEARCH Vol. 25, No. 1, 1989 Copyright O 1989 International Pediatric Research Foundation, Inc. Printed in U.S.A. The Effect of Diazepam on Neonatal Seizure: In Vivo 31Pand 'H NMR Study1 RICHARD S. K. YOUNG, BENJAMIN CHEN, OGNEN A. C. PETROFF, JOHN C. GORE, BARRETT E. COWAN, EDWARD J. NOVOTNY, JR., MABEL WONG, AND KAYE ZUCKERMAN Departments of Pediatrics [R.S.K. Y., M. W., K.Z.], Neurology [R.S.K. Y., O.A.C.P., E.J.N.], and Diagnostic Radiology [B.C., J.C.G.], Yale University School of Medicine, New Haven, Connecticut 06510-8064, and Department of Medicine [B.E.C.] Stanford University Medical School, Stanford, California 94305 ABSTRACI'. It is assumed that when anticonvulsants this study, four types of investigations were performed: in vivo arrest seizure, there is rapid return of brain high energy 31PNMR to follow brain high energy phosphate metabolism and phosphates and brain lactate to control values. To test this brain pH; in vivo 'H NMR to measure brain lactate; in vitro 'H hypothesis, diazepam was administered to neonatal dogs NMR spectroscopy to determine concentrations of brain excita- during flurothyl-induced seizure. In vivo 3'P nuclear mag- tory and inhibitory amino acids; and iodoantipyrine autoradi- netic resonance spectroscopy disclosed that diazepam ography to assess regional cerebral blood flow. quickly arrested electrographic seizure and restored brain phosphocreatine and inorganic phosphate to baseline val- ues. In contrast, in vivo 'H nuclear magnetic resonance MATERIALS AND METHODS spectroscopic measurements showed that arrest of seizure Animal preparation. The method of preparation has been with diazepam did not return brain lactate to control values. recently described (6). Mongrel dogs (4- 12 days old) were anes- The sustained increase in cerebral blood flow and pro- thetized with halothane, 1-4%, while undergoing tracheotomy longed elevation of brain lactate, acetate, valine, and suc- and femoral arterial catheterization. Pancuronium bromide (0.5 cinate in the postictal period indicate that metabolic recov- ml subcutaneously) was then administered, halothane was with- ery of the brain occurs over an extended period of time drawn, and the animals were mechanically ventilated (PaO2, 80- after the normalization of EEG, phosphocreatine, and brain 120 mm Hg; Pco2, 30-40 mm Hg). Muscle paralysis was contin- pH. (Pediatr Res 25:27-31, 1989) ued throughout the experiment. To minimize discomfort, ani- mals were ventilated with 30% 02 and 70% N20 (1, 2), and Abbreviations topical anesthetic (lidocaine jelly, 1%) was applied to all incision sites. Anesthesia during the ictus was produced by the seizure NMR, nuclear magnetic resonance itself which results in loss of awareness. The EEG, blood pressure, PCr, phosphocreatine blood glucose, and blood lactate were monitored during the pHi, intracellular pH experiment (6). Experiments were approved by the Yale Univer- CBF, cerebral blood flow sity Animal Care Committee and carried out with the adherence to "Guiding Principles for the Care of Animals," of the American Physiological Society, and in accordance with federal regulations. Animals were randomly assigned either to a continuous seizure Diazepam is widely used in the treatment of status epilepticus or to a seizure plus diazepam group. Animals in the continuous because it rapidly arrests electrographic seizure. Nonetheless, the seizure group were subjected to 60 min of seizure which was potential beneficial effects of diazepam on brain metabolism induced by continuously vaporizing flurothyl (0.13 ml/min) during the postictal period have not been fully defined in the within the ventilator tubing. Flurothyl was similarly administered neonate. Previous studies of postictal brain metabolism in the to those animals randomized to the seizure plus diazepam group, adult experimental animal (1, 2) necessitated multiple groups of but diazepam (0.67-1.0 mg/kg intravenously) was given 15 min animals at different time points (1-4). Moreover, brain pH was after onset of seizure. An additional dose of diazepam was measured by indirect methods or by invasive techniques which administered 15 min later (30 min after seizure onset) to assure themselves could perturb brain metabolism. suppression of electrographic seizure. The goal of these experiments was to use NMR spectroscopy In vivo "P NMR studies. In vivo "P data were collected using to assess rate of normalization of brain metabolism after treat- a 2.0 Tesla, 3 1-cm bore diameter superconducting magnet (Gen- ment of neonatal status epilepticus with diazepam. Seizure was eral Electric CSI 11, (CSI 11; General Electric, Fremont, CA) and induced with flurothyl (bistrifluorethyl ether; Flura Corp., New- a 2-cm circular transmitter-receiver surface coil tuned for phos- port, TN), a convulsant gas which causes seizure by opening of phorus (34.5 MHz, 512 acquisitions, repetition rate, 1.0 s). sodium channels in the cell membrane (2). Previous studies have Reflection of cranial muscle was not done because previous shown that flurothyl seizure retards brain growth in the neonatal imaging studies (6) show that most of the signal is derived from rat (5) and produces neuronal necrosis in the adult rat (4). In the relatively large brain of the neonatal dog. Moreover, the PCr/ P, ratio in the control state was near unity, indicating minimal Received May 3 1, 1988; accepted August 3 1, 1988. Correspondence Richard S.K. Young, M.D., Department of Pediatrics, Yale contamination from surrounding muscle (7). Peak areas were University School of Medicine, 333 Cedar St., New Haven, CT 065 10-8064. not corrected for differential saturation because quantitative Supported by NIH Grants NS R01-24605 (R.S.K.Y.), NS 21708 (O.A.C.P.), measurement of individual resonances was not attempted. and T32 CA09549 (B.C.), and during the tenure of a Clinician-Scientist Award Sets of 31PNMR spectra were acquired every 15 min through- from the American Heart Association (R.S.K.Y.). ' Presented in part at the Annual Meeting of the Child Neurology Society, out the experiment. The curve-fitting software "GEMCAP" (Ni- October 1987. colet Computer Graphics Div., Martinez, CA) was used to deter- 28 YOUNG ET AL. mine areas of individual resonances where peak overlap oc- the spectrometer at the end of the experiment, acquiring a curred. Brain pHi was determined from experimental spectra by spectrum 20 min postmortem, and correlating the intensity of noting the chemical shift of the Pi peak with respect to that of the lactate signal to the enzymatically derived lactate concentra- PCr. as previously described (8). tion in brain (9). In vivo 'H NMR studies. In vivo 'H NMR spectra were obtained In vitro 'H NMR studies. In vitro 'H NMR investigations were with a 133T spin echo sequence (9) using the same spectrometer performed in control animals, continuously seizing animals, and ('H frequency, 85.6 MHz; 32 scans). A separate group of animals animals whose seizures were arrested with diazepam. Brains of and a dedicated proton coil (General Electric, 2-cm circular) these animals were frozen in situ with liquid nitrogen (10) and were used to maximize signal to noise and improve temporal later dissected and extracted with HC104. Extracts were analyzed resolution. Brain lactate was quantitated by killing the animal in for metabolite concentrations by conventional spectrophotomet- ric analysis (I 1) and with high resolution 'H NMR spectroscopy (500 MHz Bruker WM-500 spectrometer; NMR parameters: Flurothvl Flurothvl - Diazepam 25"C, 30" pulse, 6-s repetition time, 160 scans). Resonances were 16 1 Blood Hflf.. assigned relative to sodium 3-trimethyl silyl proprionate (6). 14 Glucose Regional CBF. Regional CBF was measured by quantitative autoradiography ([I4C] iodoantipyrine) (12) in a parallel group of animals subjected to the same experimental conditions. CBF was determined at the end of the 60-min period of experimental observation. DlUUU ulooa 4.0 1 Lactate / 1 4.0t Lactate Statistical analyses. The need for a control group is obviated in in vivo NMR studies because animals serve as their own controls. Sequential intragroup data obtained with in vivo NMR were therefore analyzed with repeated measures analysis of var- iance ANOVA and the Newman-Keuls post hoe test. Because the in vitro NMR and CBF studies necessitated multiple groups, statistically significant (p< 0.05) differences were analyzed with randomized ANOVA. All values are mean f SE. -0 7.1 -0 Time (min.) T~me(min.) RESULTS Flurothyl Flurothyl 7 - Diazepam Systemic changes. Onset of seizure resulted in systemic arterial Fig. 1. Systemic changes during flurothyl seizure. Blood glucose rises hypertension, whereas administration of diazepam caused a to similar degree in animals subjected to continuous flurothyl seizure slight reduction in blood pressure (control, 93 + 6 mm Hg; (right) and those treated with diazepam (left). However, administration flurothyl, 108 + 8; flurothyl and diazepam, 81 +- 3; p = 0.012). of diazepam is associated with decrease in lactate. A similar degree of Flurothyl seizure caused significant increase in blood glucose, metabolic acidosis occurred in both groups of animals. Data from a total which was most prominent early in the course of seizure (Fig. of 12 animals (continuous flurothyl seizure, seven; flurothyl plus diaze- 1). Blood lactate increased in response to flurothyl seizure, but pam, five) are depicted. then plateaued once seizure was treated with diazepam. Reduc- I DIAZEPAM EEG I ATP I .,------ - / 1- Sec I SEIZURE EEG I 1 Sec CONTROL EEG Fig. 2. 31PNMR spectra and EEG during flurothyl seizure and after treatment with diazepam. The ratio of PCr to inorganic phosphate (dashed line between PCr and Pi peaks) abruptly decreases with onset of seizure and then gradually recovers. Note the rapid termination of electrographic seizure after treatment with diazepam. Abbreviations: PM, phosphomonoesters; PD, phosphodiesters; y, a, and /3 are resonances of ATP. "P AND IH NMR STUDY OF NEONATAL SEIZURE AND DIAZEPAM 29 tion in arterial pH was similar in both the continuous flurothyl ity.