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Mild postischemic hypothermia is neuroprotective in the immature rat neocortex slice
Brain Research 894 (2001) 297–300 www.elsevier.com / locate / bres
Short communication
Mild postischemic hypothermia is neuroprotective in the immature rat neocortex slice a, b b a Hae-Kyung Ko *, Melanie Zeller , Siegrun Gabriel , Johannes Graulich , Uwe Heinemann b , Michael Obladen a a
Department of Neonatology, Charite´ Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany b Johannes-Mueller-Institute of Physiology, Charite´ , Humboldt-University Berlin, Germany Accepted 31 October 2000
Abstract Mild hypothermia as an intervention after perinatal asphyxia may prevent neurological damage in the newborn. We used stimulusinduced field potentials to monitor recovery from oxygen and glucose deprivation (OGD) in neocortex slices of 6–8-day-old wistar rats. OGD after a latency of 10.762.1 min (mean6S.E.) resulted in an anoxic depolarisation with an amplitude of 5.462.4 mV. Mild hypothermia of 318C (vs. 358C in the control group) was applied for 60 min after end of OGD. The 20, 40, 60 and 80% recovery of the field potential amplitude was significantly faster in the hypothermia group in comparison to the control group. These data indicate that mild postischemic hypothermia may have neuroprotective effects after perinatal asphyxia. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Newborn; Oxygen and glucose deprivation; Neocortex; Slice; Temperature
1. Introduction Perinatal asphyxia as a cause of brain damage in newborns with subsequent longterm neurological morbidity still remains a therapeutical problem. Recent results of in vivo animal experiments revealed a therapeutic window of a few hours for interventions after the hypoxic–ischemic insult [2,3,5]. First clinical trials on mild hypothermia as a neuroprotective option after perinatal asphyxia in human newborns are being run. Preliminary results of the studies [4,16] and also retrospective analyses [11] showed that in terms of side effects low rate and moderate severity of complications may allow a hypothermic intervention in neonates endangered by severe cerebral damage. In vitro models of hypoxic–ischemic neuronal damage in brain slices allow investigations under standardized conditions and temperatures. In previous studies intraischemic hypothermia of moderate to severe degree was investigated *Corresponding author. Tel.: 149-30-45050; fax: 149-30-4506-6922. E-mail address: [email protected] (H.-K. Ko).
in the adult brain [9,13]. Hypothermia by more than 4–58C because of more severe side effects may be difficult to apply in the clinical setting and transfer of results obtained in the adult brain to the newborn brain is limited by different mechanisms of neuronal injury and a higher tolerance to hypoxia / ischemia in the newborn brain. Thus we tested the hypothesis that a mild postischemic hypothermia in vitro is neuroprotective in the immature neocortex by measuring the recovery of the stimulus-induced field potential amplitude.
2. Experimental protocol All experiments were done on coronal 400-mm somatosensory cortex slices from male and female wistar rat pups (age 6–8 days) as described previously [8]. In short, animals were decapitated under ether anesthesia and the brain was removed and stored in ice-cold artificial cerebrospinal fluid (aCSF) containing 129 mM NaCl, 1.25 mM NaH 2 PO 4 , 1.8 mM MgSO 4 , 1.6 mM CaCl 2 , 3 mM KCl,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03188-7
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H.-K. Ko et al. / Brain Research 894 (2001) 297 – 300
21 mM NaHCO 3 and 10 mM glucose, temperature about 48C, oxygenation with 95% O 2 –5% CO 2 ; glucose-free aCSF contained 5 mM additional NaCl for osmotic equilibration. Slices were cut using a vibratome (Campden Instruments, Loughborough, UK) and were allowed to recover from preparation for 60 min in an interface recording chamber under continuous oxygenation with carbogen (95% O 2 –5% CO 2 ) and perfusion with aCSF, pH 7.4, perfusion rate 1.6 ml / min. The temperature was measured at the border of the slice and regulated by the chamber heating system. Changes of temperature were made in steps of 0.158C every 2 min; thus, the changes between normothermia of 358C and hypothermia of 318C were performed within 30 min. For recordings a double-barreled calcium-sensitive microelectrode — prepared and tested as described previously [6] — was placed in layer II–III of the somatosensory cortex (depth 80 mm) and a bipolar stimulating electrode was positioned in the underlying white matter. An initial input–output profile (20–120% stimulus intensity) of the stimulus-induced field potential (FP) was obtained at 318C and was again performed after warming to 358C. Only slices with an amplitude of more than 1 mV were included into the protocol. Oxygen and glucose deprivation (OGD) was induced by switching aeration of recording chamber and aCSF from 95% O 2 –5% CO 2 to 95% N 2 –5% CO 2 and by removing glucose from the aCSF. We chose a relatively long duration of OGD of 6 min after onset of anoxic depolarisation for our experiments (average duration of OGD 16 min). Stimulus-induced FP amplitude and baseline extracellular calcium concentration were recorded every 20 s during OGD and every 10 min during postischemic recovery after OGD. Complete functional recovery was defined as 95% amplitude of the preischemic value at 80% stimulus intensity. To account for temperature-dependent changes in FP amplitude preischemic values were derived from the input–output profile at 31 and 358C, respectively. For postischemic hypothermia, slices were cooled to
318C immediately after the end of OGD and temperature was held at 318C for 60 min. Controls were performed in normothermia (358C), total observation time was 2.5 h after end of OGD. Data were printed on-line on a chart writer and stored on hard disk for further analysis. Data are presented as mean6standard error, statistical differences were tested with non-parametrical Mann–Whitney U test and Wilcoxon test for independent and dependent samples, respectively and Kruskal–Wallis test for multiple analysis of independent samples at a significance level of P,0.05 ( SPSS software package).
3. Results In normoxic and normoglycemic conditions at 318C FP amplitudes were 3.160.3 mV with 80% stimulus intensity in the hypothermia group (n513) and 3.160.1 mV in the control group (n58). At 358C they were 2.960.2 and 2.860.3 mV in hypothermia and control groups, respectively (Fig. 1A and B). FP amplitudes at 31 and 358C and at stimulus intensities of 20, 40, 60, 100 and 120% were not significantly different either within the groups (Wilcoxon test) and between the groups (Mann–Whitney U test). An OGD of 10 min resulted in a complete suppression of FP in all slices. A negative shift of the baseline field potential indicated anoxic depolarisation (AD) after a latency of 10.762.1 min with an amplitude of 5.462.4 mV (representative example in Fig. 2). In 30% of the slices an initial positive baseline shift of ,1 mV was observed before onset of the AD. Extracellular calcium concentration decreased from 1.2 to 0.260.3 mmol / l during OGD. After end of OGD both baseline field potential and extracellular calcium concentration decreased further for another 1–2 min before a slow recovery (Fig. 2). AD latencies and AD amplitudes and decreases in extracellular calcium concentration did not significantly differ in the hypothermia group as compared to the control group
Fig. 1. Field potential (FP) recorded in neocortex layer II / III in response to stimulation of underlying white matter. (A) Typical FP recording (*; 80% stimulus intensity). Arrow represents determination of amplitude. (B) Effects of temperature on FP amplitudes at 31 and 358C for hypothermia and control groups (mean6S.E.). Differences in amplitudes were not significant.
H.-K. Ko et al. / Brain Research 894 (2001) 297 – 300
Fig. 2. Changes of baseline field potential (FP) and extracellular calcium concentration [Ca 21 ] o during oxygen and glucose deprivation (OGD) in on-line registration. OGD was started 11 min before onset of anoxic depolarisation (AD) and was terminated as indicated by arrow. There were no significant differences in AD latency and amplitude and changes of [Ca 21 ] o in the hypothermia group as compared to the control group.
(Mann–Whitney U test). The time of recovery to 20% of the preischemic value was determined and was 16.265.2 min in the hypothermia group in comparison to 24.363.1 min in the control group. Likewise 40, 60 and 80% of the preischemic value were reached in 28.864.4, 42.565.3 and 58.864.8 min in the hypothermia group versus 4566.0, 65.567.1 and 68.864.0 min in the control group (Fig. 3). These differences were statistically significant (multiple analysis by Kruskal–Wallis test). After the end of the observation time of 2.5 h 63% (seven out of thirteen) of the slices in the hypothermia group and 38% (three out of eight) of the slices in the control group reached 100% of the preischemic amplitude.
4. Discussion This is the first in vitro study to investigate the neuroprotective effect of mild hypothermia induced after OGD in the immature brain judged by recovery of stimulus-
Fig. 3. Recovery of field potential (FP) amplitude after oxygen and glucose deprivation (OGD). Amplitudes are given in percent of preischemic value derived with respect to recording temperatures of 31 and 358C, respectively, (abscissa) and in dependence of time after OGD (ordinate). *, P,0.05.
299
induced field potentials. Our main finding was an improved recovery of field potentials in the hypothermia group as compared to the control group suggesting that hypothermia by 48C might be neuroprotective in the immature brain. The neuroprotective effect we found is particularly impressing considering the relatively long OGD period of about 16 min with occurrence of AD in all slices. In preceeding in vitro investigations the duration of hypoxia or OGD was shorter. Moreover, in immature brain slices hypoxia alone induced AD in only a part of the slices [8]. Also the latency to hypothermia of 30 min was longer than in every other in vitro study, although it was still shorter than in some in vivo animal investigations [5] in which the therapeutical window was shown to be up to 6 h with still having neuroprotective effects. In preliminary clinical studies an interval of 6 h after delivery has also proven to be realistic for preintervention procedures like clinical assessment for indication to treat and preparation of technical facilities [4,11]. To aim for a better transfer of data from in vitro investigations to the clinical setting further studies are therefore needed that include an extention of the postischemic interval before beginning of neuroprotective interventions. Our findings are at variance with studies in adult animals where temperature changes were performed more rapidly [15,17]. We chose a slow cooling protocol as in the clinical setting a change of core temperature as therapeutic intervention would likewise take rather long and cannot be performed within a few minutes. We did not find a consistent decrease in FP amplitudes with warming of the slices. This may as well be explained by the slow temperature changes performed in our protocol. In previous investigations temperature changes in the recording chamber were performed at about 18C / min [10] in contrast to less than 0.18C / min in our study. In addition there may be possibly no significant effect of temperature on FP amplitudes within the temperature range investigated (but see also [10,14]. We did not study moderate to severe hypothermia (below 308C) because of the higher risk of severe side effects to be expected in the human newborn. We followed recovery of field potentials longer than in every other in vitro study known to us and we found a greater percentage of complete recovery in the hypothermia group as compared to the control group. From our data we cannot state anything about neuronal loss and correlation to field potential. Previous studies have shown in hippocampal slice cultures of neonatal rats as well as in acute neocortex slices of adult rats that field potential amplitude correlated with cell death judged by propidium iodide and trypane blue staining, respectively [7,12]. Dooley and Corbett have shown in a long term in vivo study in the gerbil that in dependence of the time point of postischemic investigation field potential recovery does not necessarily have to be correlated to neuronal death judged by histological criteria.
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H.-K. Ko et al. / Brain Research 894 (2001) 297 – 300
They found that 3 and 10 days after in vivo ischemia field potential amplitude was still depressed whereas morphological assessment revealed a good neuronal preservation. After 90 days both field potentials as well as neuronal integrity were markedly impaired [1]. Mild postischemic hypothermia may be a promising intervention after birth asphyxia in the newborn although in the presented in vitro investigation no statement can be made about long-term effects of neuroprotection. Further studies are now needed to show whether functional recovery is also associated with preservation of neurons.
References [1] P. Dooley, D. Corbett, Competing processes of cell death and recovery of function following ischemic preconditioning, Brain Res. 794 (1998) 119–126. [2] A.D. Edwards, X. Yue, M.V. Squier, M. Thoresen, E.B. Cady, J. Penrice, C.E. Cooper, J.S. Wyatt, E.O. Reynolds, H. Mehmet, Specific inhibition of apoptosis after cerebral hypoxia–ischaemia by moderate post-insult hypothermia, Biochem. Biophys. Res. Commun. 217 (1995) 1193–1199. [3] A.J. Gunn, L. Bennet, M.I. Gunning, P.D. Gluckman, T.R. Gunn, Cerebral hypothermia is not neuroprotective when started after postischemic seizures in fetal sheep, Pediatr. Res. 46 (1999) 274– 280. [4] A.J. Gunn, P.D. Gluckman, T.R. Gunn, Selective head cooling in newborn infants after perinatal asphyxia: a safety study [see comments], Pediatrics 102 (1998) 885–892. [5] A.J. Gunn, T.R. Gunn, M.I. Gunning, C.E. Williams, P.D. Gluckman, Neuroprotection with prolonged head cooling started before postischemic seizures in fetal sheep, Pediatrics 102 (1998) 1098– 1106. [6] U. Heinemann, H.D. Lux, M.J. Gutnick, Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain. Res. 27 (1977) 237–243.
[7] R. Kovacs, R. Gutierrez, A. Kivi, S. Schuchmann, S. Gabriel, U. Heinemann, Acute cell damage after low Mg 21 -induced epileptiform activity in organotypic hippocampal slice cultures, Neuroreport 10 (1999) 207–213. [8] H.J. Luhmann, T. Kral, Hypoxia-induced dysfunction in developing rat neocortex, J. Neurophysiol. 78 (1997) 1212–1221. [9] Y. Okada, M. Tanimoto, K. Yoneda, The protective effect of hypothermia on reversibility in the neuronal function of the hippocampal slice during long lasting anoxia, Neurosci. Lett. 84 (1988) 277–282. [10] K.F. Shen, P.A. Schwartzkroin, Effects of temperature alterations on population and cellular activities in hippocampal slices from mature and immature rabbit, Brain Res. 475 (1988) 305–316. [11] G. Simbruner, C. Haberl, V. Harrison, L. Linley, A.E. Willeitner, Induced brain hypothermia in asphyxiated human newborn infants: a retrospective chart analysis of physiological and adverse effects, Intens. Care. Med. 25 (1999) 1111–1117. [12] A. Siniscalchi, C. Zona, G. Sancesario, E. D’Angelo, Y.C. Zeng, N.B. Mercuri, G. Bernardi, Neuroprotective effects of riluzole: an electrophysiological and histological analysis in an in vitro model of ischemia, Synapse 32 (1999) 147–152. [13] T. Takata, M. Nabetani, Y. Okada, Effects of hypothermia on the neuronal activity, [Ca 21 ] i accumulation and ATP levels during oxygen and / or glucose deprivation in hippocampal slices of guinea pigs, Neurosci. Lett. 227 (1997) 41–44. [14] M. Tanimoto, Y. Okada, The protective effect of hypothermia on hippocampal slices from guinea pig during deprivation of oxygen and glucose, Brain Res. 417 (1987) 239–246. [15] E. Tasdemiroglu, Mild hypothermia fails to protect late hippocampal neuronal loss following forebrain cerebral ischaemia in rats, Acta Neurochir. (Wien) 138 (1996) 570–578. [16] M. Thoresen, A. Whitelaw, Cardiovascular changes during mild therapeutic hypothermia and rewarming in infants with hypoxic– ischemic encephalopathy, Pediatrics 106 (2000) 92–99. [17] F.A. Welsh, V.A. Harris, Postischemic hypothermia fails to reduce ischemic injury in gerbil hippocampus, J. Cereb. Blood Flow Metab. 11 (1991) 617–620.
Short communication
Mild postischemic hypothermia is neuroprotective in the immature rat neocortex slice a, b b a Hae-Kyung Ko *, Melanie Zeller , Siegrun Gabriel , Johannes Graulich , Uwe Heinemann b , Michael Obladen a a
Department of Neonatology, Charite´ Campus Virchow-Klinikum, Augustenburger Platz 1, D-13353 Berlin, Germany b Johannes-Mueller-Institute of Physiology, Charite´ , Humboldt-University Berlin, Germany Accepted 31 October 2000
Abstract Mild hypothermia as an intervention after perinatal asphyxia may prevent neurological damage in the newborn. We used stimulusinduced field potentials to monitor recovery from oxygen and glucose deprivation (OGD) in neocortex slices of 6–8-day-old wistar rats. OGD after a latency of 10.762.1 min (mean6S.E.) resulted in an anoxic depolarisation with an amplitude of 5.462.4 mV. Mild hypothermia of 318C (vs. 358C in the control group) was applied for 60 min after end of OGD. The 20, 40, 60 and 80% recovery of the field potential amplitude was significantly faster in the hypothermia group in comparison to the control group. These data indicate that mild postischemic hypothermia may have neuroprotective effects after perinatal asphyxia. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Ischemia Keywords: Newborn; Oxygen and glucose deprivation; Neocortex; Slice; Temperature
1. Introduction Perinatal asphyxia as a cause of brain damage in newborns with subsequent longterm neurological morbidity still remains a therapeutical problem. Recent results of in vivo animal experiments revealed a therapeutic window of a few hours for interventions after the hypoxic–ischemic insult [2,3,5]. First clinical trials on mild hypothermia as a neuroprotective option after perinatal asphyxia in human newborns are being run. Preliminary results of the studies [4,16] and also retrospective analyses [11] showed that in terms of side effects low rate and moderate severity of complications may allow a hypothermic intervention in neonates endangered by severe cerebral damage. In vitro models of hypoxic–ischemic neuronal damage in brain slices allow investigations under standardized conditions and temperatures. In previous studies intraischemic hypothermia of moderate to severe degree was investigated *Corresponding author. Tel.: 149-30-45050; fax: 149-30-4506-6922. E-mail address: [email protected] (H.-K. Ko).
in the adult brain [9,13]. Hypothermia by more than 4–58C because of more severe side effects may be difficult to apply in the clinical setting and transfer of results obtained in the adult brain to the newborn brain is limited by different mechanisms of neuronal injury and a higher tolerance to hypoxia / ischemia in the newborn brain. Thus we tested the hypothesis that a mild postischemic hypothermia in vitro is neuroprotective in the immature neocortex by measuring the recovery of the stimulus-induced field potential amplitude.
2. Experimental protocol All experiments were done on coronal 400-mm somatosensory cortex slices from male and female wistar rat pups (age 6–8 days) as described previously [8]. In short, animals were decapitated under ether anesthesia and the brain was removed and stored in ice-cold artificial cerebrospinal fluid (aCSF) containing 129 mM NaCl, 1.25 mM NaH 2 PO 4 , 1.8 mM MgSO 4 , 1.6 mM CaCl 2 , 3 mM KCl,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )03188-7
298
H.-K. Ko et al. / Brain Research 894 (2001) 297 – 300
21 mM NaHCO 3 and 10 mM glucose, temperature about 48C, oxygenation with 95% O 2 –5% CO 2 ; glucose-free aCSF contained 5 mM additional NaCl for osmotic equilibration. Slices were cut using a vibratome (Campden Instruments, Loughborough, UK) and were allowed to recover from preparation for 60 min in an interface recording chamber under continuous oxygenation with carbogen (95% O 2 –5% CO 2 ) and perfusion with aCSF, pH 7.4, perfusion rate 1.6 ml / min. The temperature was measured at the border of the slice and regulated by the chamber heating system. Changes of temperature were made in steps of 0.158C every 2 min; thus, the changes between normothermia of 358C and hypothermia of 318C were performed within 30 min. For recordings a double-barreled calcium-sensitive microelectrode — prepared and tested as described previously [6] — was placed in layer II–III of the somatosensory cortex (depth 80 mm) and a bipolar stimulating electrode was positioned in the underlying white matter. An initial input–output profile (20–120% stimulus intensity) of the stimulus-induced field potential (FP) was obtained at 318C and was again performed after warming to 358C. Only slices with an amplitude of more than 1 mV were included into the protocol. Oxygen and glucose deprivation (OGD) was induced by switching aeration of recording chamber and aCSF from 95% O 2 –5% CO 2 to 95% N 2 –5% CO 2 and by removing glucose from the aCSF. We chose a relatively long duration of OGD of 6 min after onset of anoxic depolarisation for our experiments (average duration of OGD 16 min). Stimulus-induced FP amplitude and baseline extracellular calcium concentration were recorded every 20 s during OGD and every 10 min during postischemic recovery after OGD. Complete functional recovery was defined as 95% amplitude of the preischemic value at 80% stimulus intensity. To account for temperature-dependent changes in FP amplitude preischemic values were derived from the input–output profile at 31 and 358C, respectively. For postischemic hypothermia, slices were cooled to
318C immediately after the end of OGD and temperature was held at 318C for 60 min. Controls were performed in normothermia (358C), total observation time was 2.5 h after end of OGD. Data were printed on-line on a chart writer and stored on hard disk for further analysis. Data are presented as mean6standard error, statistical differences were tested with non-parametrical Mann–Whitney U test and Wilcoxon test for independent and dependent samples, respectively and Kruskal–Wallis test for multiple analysis of independent samples at a significance level of P,0.05 ( SPSS software package).
3. Results In normoxic and normoglycemic conditions at 318C FP amplitudes were 3.160.3 mV with 80% stimulus intensity in the hypothermia group (n513) and 3.160.1 mV in the control group (n58). At 358C they were 2.960.2 and 2.860.3 mV in hypothermia and control groups, respectively (Fig. 1A and B). FP amplitudes at 31 and 358C and at stimulus intensities of 20, 40, 60, 100 and 120% were not significantly different either within the groups (Wilcoxon test) and between the groups (Mann–Whitney U test). An OGD of 10 min resulted in a complete suppression of FP in all slices. A negative shift of the baseline field potential indicated anoxic depolarisation (AD) after a latency of 10.762.1 min with an amplitude of 5.462.4 mV (representative example in Fig. 2). In 30% of the slices an initial positive baseline shift of ,1 mV was observed before onset of the AD. Extracellular calcium concentration decreased from 1.2 to 0.260.3 mmol / l during OGD. After end of OGD both baseline field potential and extracellular calcium concentration decreased further for another 1–2 min before a slow recovery (Fig. 2). AD latencies and AD amplitudes and decreases in extracellular calcium concentration did not significantly differ in the hypothermia group as compared to the control group
Fig. 1. Field potential (FP) recorded in neocortex layer II / III in response to stimulation of underlying white matter. (A) Typical FP recording (*; 80% stimulus intensity). Arrow represents determination of amplitude. (B) Effects of temperature on FP amplitudes at 31 and 358C for hypothermia and control groups (mean6S.E.). Differences in amplitudes were not significant.
H.-K. Ko et al. / Brain Research 894 (2001) 297 – 300
Fig. 2. Changes of baseline field potential (FP) and extracellular calcium concentration [Ca 21 ] o during oxygen and glucose deprivation (OGD) in on-line registration. OGD was started 11 min before onset of anoxic depolarisation (AD) and was terminated as indicated by arrow. There were no significant differences in AD latency and amplitude and changes of [Ca 21 ] o in the hypothermia group as compared to the control group.
(Mann–Whitney U test). The time of recovery to 20% of the preischemic value was determined and was 16.265.2 min in the hypothermia group in comparison to 24.363.1 min in the control group. Likewise 40, 60 and 80% of the preischemic value were reached in 28.864.4, 42.565.3 and 58.864.8 min in the hypothermia group versus 4566.0, 65.567.1 and 68.864.0 min in the control group (Fig. 3). These differences were statistically significant (multiple analysis by Kruskal–Wallis test). After the end of the observation time of 2.5 h 63% (seven out of thirteen) of the slices in the hypothermia group and 38% (three out of eight) of the slices in the control group reached 100% of the preischemic amplitude.
4. Discussion This is the first in vitro study to investigate the neuroprotective effect of mild hypothermia induced after OGD in the immature brain judged by recovery of stimulus-
Fig. 3. Recovery of field potential (FP) amplitude after oxygen and glucose deprivation (OGD). Amplitudes are given in percent of preischemic value derived with respect to recording temperatures of 31 and 358C, respectively, (abscissa) and in dependence of time after OGD (ordinate). *, P,0.05.
299
induced field potentials. Our main finding was an improved recovery of field potentials in the hypothermia group as compared to the control group suggesting that hypothermia by 48C might be neuroprotective in the immature brain. The neuroprotective effect we found is particularly impressing considering the relatively long OGD period of about 16 min with occurrence of AD in all slices. In preceeding in vitro investigations the duration of hypoxia or OGD was shorter. Moreover, in immature brain slices hypoxia alone induced AD in only a part of the slices [8]. Also the latency to hypothermia of 30 min was longer than in every other in vitro study, although it was still shorter than in some in vivo animal investigations [5] in which the therapeutical window was shown to be up to 6 h with still having neuroprotective effects. In preliminary clinical studies an interval of 6 h after delivery has also proven to be realistic for preintervention procedures like clinical assessment for indication to treat and preparation of technical facilities [4,11]. To aim for a better transfer of data from in vitro investigations to the clinical setting further studies are therefore needed that include an extention of the postischemic interval before beginning of neuroprotective interventions. Our findings are at variance with studies in adult animals where temperature changes were performed more rapidly [15,17]. We chose a slow cooling protocol as in the clinical setting a change of core temperature as therapeutic intervention would likewise take rather long and cannot be performed within a few minutes. We did not find a consistent decrease in FP amplitudes with warming of the slices. This may as well be explained by the slow temperature changes performed in our protocol. In previous investigations temperature changes in the recording chamber were performed at about 18C / min [10] in contrast to less than 0.18C / min in our study. In addition there may be possibly no significant effect of temperature on FP amplitudes within the temperature range investigated (but see also [10,14]. We did not study moderate to severe hypothermia (below 308C) because of the higher risk of severe side effects to be expected in the human newborn. We followed recovery of field potentials longer than in every other in vitro study known to us and we found a greater percentage of complete recovery in the hypothermia group as compared to the control group. From our data we cannot state anything about neuronal loss and correlation to field potential. Previous studies have shown in hippocampal slice cultures of neonatal rats as well as in acute neocortex slices of adult rats that field potential amplitude correlated with cell death judged by propidium iodide and trypane blue staining, respectively [7,12]. Dooley and Corbett have shown in a long term in vivo study in the gerbil that in dependence of the time point of postischemic investigation field potential recovery does not necessarily have to be correlated to neuronal death judged by histological criteria.
300
H.-K. Ko et al. / Brain Research 894 (2001) 297 – 300
They found that 3 and 10 days after in vivo ischemia field potential amplitude was still depressed whereas morphological assessment revealed a good neuronal preservation. After 90 days both field potentials as well as neuronal integrity were markedly impaired [1]. Mild postischemic hypothermia may be a promising intervention after birth asphyxia in the newborn although in the presented in vitro investigation no statement can be made about long-term effects of neuroprotection. Further studies are now needed to show whether functional recovery is also associated with preservation of neurons.
References [1] P. Dooley, D. Corbett, Competing processes of cell death and recovery of function following ischemic preconditioning, Brain Res. 794 (1998) 119–126. [2] A.D. Edwards, X. Yue, M.V. Squier, M. Thoresen, E.B. Cady, J. Penrice, C.E. Cooper, J.S. Wyatt, E.O. Reynolds, H. Mehmet, Specific inhibition of apoptosis after cerebral hypoxia–ischaemia by moderate post-insult hypothermia, Biochem. Biophys. Res. Commun. 217 (1995) 1193–1199. [3] A.J. Gunn, L. Bennet, M.I. Gunning, P.D. Gluckman, T.R. Gunn, Cerebral hypothermia is not neuroprotective when started after postischemic seizures in fetal sheep, Pediatr. Res. 46 (1999) 274– 280. [4] A.J. Gunn, P.D. Gluckman, T.R. Gunn, Selective head cooling in newborn infants after perinatal asphyxia: a safety study [see comments], Pediatrics 102 (1998) 885–892. [5] A.J. Gunn, T.R. Gunn, M.I. Gunning, C.E. Williams, P.D. Gluckman, Neuroprotection with prolonged head cooling started before postischemic seizures in fetal sheep, Pediatrics 102 (1998) 1098– 1106. [6] U. Heinemann, H.D. Lux, M.J. Gutnick, Extracellular free calcium and potassium during paroxysmal activity in the cerebral cortex of the cat, Exp. Brain. Res. 27 (1977) 237–243.
[7] R. Kovacs, R. Gutierrez, A. Kivi, S. Schuchmann, S. Gabriel, U. Heinemann, Acute cell damage after low Mg 21 -induced epileptiform activity in organotypic hippocampal slice cultures, Neuroreport 10 (1999) 207–213. [8] H.J. Luhmann, T. Kral, Hypoxia-induced dysfunction in developing rat neocortex, J. Neurophysiol. 78 (1997) 1212–1221. [9] Y. Okada, M. Tanimoto, K. Yoneda, The protective effect of hypothermia on reversibility in the neuronal function of the hippocampal slice during long lasting anoxia, Neurosci. Lett. 84 (1988) 277–282. [10] K.F. Shen, P.A. Schwartzkroin, Effects of temperature alterations on population and cellular activities in hippocampal slices from mature and immature rabbit, Brain Res. 475 (1988) 305–316. [11] G. Simbruner, C. Haberl, V. Harrison, L. Linley, A.E. Willeitner, Induced brain hypothermia in asphyxiated human newborn infants: a retrospective chart analysis of physiological and adverse effects, Intens. Care. Med. 25 (1999) 1111–1117. [12] A. Siniscalchi, C. Zona, G. Sancesario, E. D’Angelo, Y.C. Zeng, N.B. Mercuri, G. Bernardi, Neuroprotective effects of riluzole: an electrophysiological and histological analysis in an in vitro model of ischemia, Synapse 32 (1999) 147–152. [13] T. Takata, M. Nabetani, Y. Okada, Effects of hypothermia on the neuronal activity, [Ca 21 ] i accumulation and ATP levels during oxygen and / or glucose deprivation in hippocampal slices of guinea pigs, Neurosci. Lett. 227 (1997) 41–44. [14] M. Tanimoto, Y. Okada, The protective effect of hypothermia on hippocampal slices from guinea pig during deprivation of oxygen and glucose, Brain Res. 417 (1987) 239–246. [15] E. Tasdemiroglu, Mild hypothermia fails to protect late hippocampal neuronal loss following forebrain cerebral ischaemia in rats, Acta Neurochir. (Wien) 138 (1996) 570–578. [16] M. Thoresen, A. Whitelaw, Cardiovascular changes during mild therapeutic hypothermia and rewarming in infants with hypoxic– ischemic encephalopathy, Pediatrics 106 (2000) 92–99. [17] F.A. Welsh, V.A. Harris, Postischemic hypothermia fails to reduce ischemic injury in gerbil hippocampus, J. Cereb. Blood Flow Metab. 11 (1991) 617–620.