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Available online at www.sciencedirect.com
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Research Report
Hypothermia did not prevent epilepsy following experimental status epilepticus Mirja Steinbrennera,1, Alexander B. Kowskia,1, Friedhelm C. Schmittb, Martin Holtkampa,n a Epilepsy-Center Berlin-Brandenburg, Department of Neurology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany b Department of Neurology, Universitätsklinik Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
art i cle i nfo
ab st rac t
Article history:
In epilepsy research, one of the major challenges is to prevent or at least mitigate
Accepted 12 May 2014
development of epilepsy following acquired brain insult by early therapeutic interventions. So far, all pharmacological antiepileptogenic treatment approaches were largely unsuccess-
Keywords:
ful in clinical trials and in experimental animal studies. In a well-established rat model
Brain insult
of chronic epilepsy following self-sustaining status epilepticus (SSSE), we assessed the
Cooling
antiepileptogenic properties of 3-h-cooling induced directly after the end of SSSE. Occur-
Excitation
rence of spontaneous seizures and seizure severity up to 8 weeks after SSSE were compared
Inhibition
with normothermic SSSE controls. Furthermore, electrophysiological parameters assessing
Spontaneous seizure
inhibition and excitation in the dentate gyrus were assessed at multiple time points. Post SSSE hypothermia did not prevent the occurrence of seizures in any animal. Eight weeks after SSSE, Racine motor seizures trended to be less severe following cooling (4.070.6) compared with normothermic controls (4.870.2) but the difference was not significant when testing for multiple comparisons. Early loss of inhibition that is typically seen following SSSE was somewhat attenuated in cooled animals 3 h after SSSE as expressed by smaller pairedpulse ratios (PPR; 0.1670.21) compared with normothermic controls (0.5470.21) but difference was not significant either. Latency between stimulus artefact and excitatory post-synaptic potential 3 h after SSSE, reciprocally reflecting neuronal excitation, was higher in animals that underwent hypothermia (8.2972.45 ms)
compared with controls
(4.8270.66 ms), difference was not significant after correction for multiple comparisons. In summary, the current experiments were not able to demonstrate prevention or mitigation of epileptogenesis with immediate short-term cooling following SSSE. & 2014 Published by Elsevier B.V.
n
Corresponding author. Fax: þ49 30 450 560 932. E-mail addresses: [email protected] (M. Steinbrenner), [email protected] (A.B. Kowski), [email protected] (F.C. Schmitt), [email protected] (M. Holtkamp). 1 MS and ABK contributed equally to this manuscript. http://dx.doi.org/10.1016/j.brainres.2014.05.018 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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1.
Introduction
Antiepileptic treatment commonly refers to suppression of unprovoked epileptic seizures in established epilepsy that in fact represents an antiictal treatment strategy. In patients with acquired brain insults such as traumatic brain injury or status epilepticus, which imply a high risk of subsequent epilepsy, early therapeutic interventions aim to completely prevent or at least mitigate the development of epilepsy representing an antiepileptogenic approach (Pitkanen and Lukasiuk, 2011). Antiepileptic drugs that are successful antiictally and a plethora of other pharmacological agents with various mechanisms of action have been assessed regarding their antiepileptogenic properties in a few clinical trials and manifoldly in animal models of epileptogenesis induced by diverse brain insults. In summary, none of these studies demonstrated an antiepileptogenic effect (Holtkamp and Meierkord, 2007; Kobow et al., 2012; Loscher and Brandt, 2010). Hypothermia, a non-pharmological treatment approach, is well-known to reduce cortical excitability. Thus, it has been applied in patients to suppress circumscribed intraoperative spiking in epilepsy surgery (Karkar et al., 2002) and to contribute to management of refractory status epilepticus (Guilliams et al., 2013; Orlowski et al., 1984). In an experimental model of electrically induced self-sustaining status epilepticus (SSSE), our group has demonstrated strong anticonvulsant effects of moderate (30 1C) and deep (20 1C) hypothermia eventually terminating continuous seizure activity (Kowski et al., 2012; Schmitt et al., 2006). In animal models of fluid percussion injury, seizure susceptibility was attenuated by early hypothermia (Atkins et al., 2010) and epileptogenesis was largely prevented by mildly lowering perilesional temperature by 2 1C for 5 weeks (D’Ambrosio et al., 2013). In the latter study, interventional hypothermia was initiated 3 d after traumatic brain injury. Relevant pathophysiological changes promoting epileptogenesis such as accumulation of N-methyl-D-aspartate (NMDA) receptors (McNamara et al., 2006) have been described to occur in the first hour after experimental status epilepticus (Naylor
et al., 2013). Due to some antiglutamatergic effects of cooling (Van Hemelrijck et al., 2003; Winfree et al., 1996), we hypothesized that induction of hypothermia for 3 h directly after termination of SSSE has antiepileptogenic properties. At various time points up to 8 weeks following SSSE, we assessed occurrence and severity of spontaneous seizures by intermittent video recording and dentate gyrus (DG) inhibition and excitation by in vivo electrophysiological measurements (for overview, see Fig. 1).
2.
Results
2.1.
Self-sustaining status epilepticus
In 23 animals, the perforant path was stimulated electrically for 2 h resulting in SSSE in 21 animals. The remainder did not fulfill our inclusion criteria as regularly occurring spontaneous discharges (Z 1 Hz) at the end of stimulation were lacking. These 21 animals maintained SSSE for the next 3 h. Two animals died directly after pentobarbital-induced termination of SSSE. Thus, a total of 19 animals were eligible for post-SSSE hypothermia or normothermia and for subsequent video recording and electrophysiological studies. In the electrode control group, nine animals were injected intraperitoneally (i.p.) pentobarbital at an index time point without prior induction of SSSE.
2.2.
Induction of hypothermia
Five minutes after i.p. administration of 30 mg/kg pentobarbital, SSSE animals either underwent cooling down to 25 1C (n ¼9) or were maintained at 37 1C core temperature (normothermic controls, n¼ 10) for 3 h. Electrode controls (n¼ 9) were kept at 37 1C for 5 h after administration of pentobarbital. In cooled animals, the target temperature was reached after 40713 min. At the end of induced hypothermia, this group of rats was rewarmed to 37 1C over another 2 h. Fig. 2 illustrates the course of body temperature in both groups of animals following SSSE.
Fig. 1 – Study overview. Animals of all three groups underwent implantation of electrodes into the right perforant path and the ipsilateral dentate gyrus. Two groups underwent electrical induction of self-sustaining status epilepticus (SSSE) that after 3 h was terminated with pentobarbital, while animals of the electrode control group remained unstimulated but were also administered pentobarbital. After the end of SSSE, one of the two groups underwent immediate cooling to 25 1C body temperature that was maintained for 3 h. The other post-SSSE group and the electrode controls were kept at 37 1C. Animals of all groups were video-monitored for periods of 48 h to detect behavioral seizures 1, 2, 4 and 8 weeks after SSSE. All animals underwent electrophysiological (e’ph) measurements to assess dentate gyrus inhibition and excitation 3 h, 24 h, 4 d, 6 d, 8 d, 2 weeks, 4 weeks and 8 weeks after SSSE. Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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2.3.
Spontaneous seizures
detected seizures without behavioral changes at weeks 2 and 8. Frequency distributions of animals with spontaneous seizures between SSSE groups were not significantly different at any time point assessed (table 1). In epileptic animals, mean number of seizures in all epochs of 48-h-video recording was 2.472.8 without any significant differences between groups at all and at the four defined time points (data not shown). Mean duration of motor seizures was 30.775.9 s in animals that underwent cooling and 32.577.1 s in normothermic controls, the difference was not significant. Motor seizure severity was assessed by the Racine scale. We calculated the mean score in each animal at each time point. Eight weeks post SSSE, animals that underwent cooling trended to lower scores (4.070.6; n¼ 6) compared with normothermic controls (4.870.2; n¼6) but difference was not significant when taking multiple comparisons into account. At all other time points assessed, we were not able to demonstrate statistically significant differences in seizure severity either (table 1).
One, 2, 4 and 8 weeks after SSSE or exclusive pentobarbital injection, animals underwent 48-h-periods of continuous video-monitoring. None of the animals in the electrode control group developed spontaneous seizures at any time point. In animals that underwent hypothermia, one rat died 2 weeks after SSSE, without any seizures detected in videorecordings in week 1 or 2. Another animal died after video recording 4 weeks post SSSE but had spontaneous seizures before. Eventually eight out of eight eligible rats were epileptic at the end of follow-up, i.e. they exhibited spontaneous seizures at least at one time point assessed. In normothermic SSSE animals, two died during 8-week-follow-up, both had seizures before. At the end of the study, nine out of 10 animals were epileptic. In the single normothermic rat without Racine stage 3 to 5 motor seizures, EEG monitoring
2.4.
Electrophysiological findings
2.4.1.
GABAergic inhibition
Prior to SSSE, small paired-pulse ratios (PPR) at short interpulse intervals (IPI) of 20 ms (0.0370.12), 25 ms (0.0370.08) and 30 ms (0.0770.14) in animals of all three groups indicated strong physiological inhibition in the DG. Typically, an IPI of 100 ms resulted in facilitation and IPIs between 300 and 1000 ms in weak inhibition. At any IPI, there was no significant difference between all three groups (data not shown). In the electrode control group, PPR as compared to baseline (the data collected before the intervention) was not significantly altered at the majority of time points assessed after administration of pentobarbital. Only at the time point 3 h after SSSE and at an IPI of 50 ms, PPR was significantly decreased as compared with pre-SSSE baseline (p¼ 0.001). As pentobarbital was administered in electrode controls and in the two post-SSSE groups of animals, possible electrophysiological differences in hypothermic animals compared with other groups can be attributed to cooling. In the 8 weeks following SSSE, in normothermic animals the changes of PPR at IPIs of 20–30 ms indicate an initial relevant loss of inhibition with a maximum 24 h after the end of status epilepticus. Impaired inhibition sustained for the
Fig. 2 – Course of body temperature. Body core temperature of experimental animals measured epidurally. Hours 0–2 indicate electrical stimulation of the dentate gyrus, hours 2–5 indicate self-sustaining status epilepticus (SSSE). Baseline temperature is approximately 37 1C. During 2-helectrial stimulation, animals exhibit numerous motor seizures (Racine class 4 and 5, i.e. rearing and falling) resulting in increased body temperature of 38–39 1C. Animals that undergo hypothermia (grey line) after termination of SSSE, reach the target temperature of 25 1C within 40713 min. Three hours after onset of cooling, animals are rewarmed to baseline temperature. Body temperature of animals in the normothermic group (black line) is kept constantly at 37 1C by a relais-controlled heating lamp.
Table 1 – Seizure occurrence and severity. Week
1 2 4 8
Animals with seizures(n/n)
Racine score(mean7SD)
Hypothermic group
Normothermic group
p-values
0/9 1/9 5/8 6/7
0/10 2/10 5/9 6/8
n.a. 0.542 0.581 0.554
n
Hypothermic group
Normothermic group
p-valuesnn
n.a. 4.070.0 4.370.6 4.070.6
n.a. 4.370.4 4.470.5 4.870.2
n.a. 0.667 0.792 0.026
n, number; SD, standard deviation; n.a., not applicable. Fischer's exact test. nn Mann–Whitney-U test, 2-sided, following Holms-Bonferroni test to correct for multiple comparisons differences are significant with po0.012. n
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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Fig. 3 – Course of inhibition following self-sustaining status epilepticus. (A) Following self-sustaining status epilepticus (SSSE), strong inhibition as seen at baseline is dramatically impaired already 3 h after termination of status epilepticus reaching its maximum at 24 h post as indicated by significantly increased paired-pulse ratios (PPR). In the following weeks, PPRs are gradually restored, baseline values are achieved 8 weeks after SSSE. Pre, before SSSE; h, hour; d, day; w, week; * po0.006. (B) Example of excitatory post-synaptic potentials and population spikes in response to conditioning and test stimuli at an interpulse interval of 20 ms before and at eight time points after SSSE. next 4 weeks and recovers to baseline 8 weeks after SSSE (Fig. 3a). An example of field potentials in response to paired stimuli at an IPI of 20 ms in the course of 8 weeks after SSSE in a normothermic animal is given in Fig. 3b.
In animals that underwent post-SSSE hypothermia, the very early loss of GABAergic inhibition 3 h after the end of SSSE trended to be mitigated. At an IPI of 25 ms, increase of PPR was less pronounced when comparing SSSE animals with
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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cooling (0.1670.21) to those with normothermia (0.5470.21; p¼ 0.027; Fig. 4a). After testing for multiple comparisons, the difference did not reach a significant level. While normothermic SSSE animals had significantly higher PPRs than electrode controls (0.070.0; p ¼0.001) indicating relevant loss of inhibition, this difference was compensated for by hypothermia (p¼ 0.083). This indirectly suggests that cooling diminished the loss of inhibition 3 h after the end of SSSE.
2.4.2.
Excitatory parameter
Analogue to the inhibitory data, latency between the stimulus and the negative peak of the population spike prior to SSSE was not significantly different between all three groups (3.6070.47 ms in all animals). In electrode controls, latency remained unchanged within the 8 weeks following administration of pentobarbital (data not shown) excluding any impact of the sedative agent. Following SSSE, in normothermic animals latency was significantly longer at one time point after SSSE (24 h) as compared with baseline (p ¼ 0.003). Cooling to 25 1C for 3 h after SSSE resulted in a trend towards longer latency comparing SSSE animals with cooling (8.2972.45 ms) to those with normothermia (4.8270.66 ms; p¼ 0.014) (Fig. 4b). This would indicate reduced neuronal excitation, but the difference was not significant when testing for multiple comparisons. While latency in normotermic SSSE animals did not differ from electrode controls (4.0570.51), latency in hypothermic SSSE animals was significant longer compared with electrode controls (p ¼0.003). This indirectly suggests that 3 h after the end of SSSE cooling results in prolonged latency and thus decreased neuronal excitation.
3.
5
Discussion
We tested the hypothesis if hypothermia applied to rats f or 3 h directly following termination of SSSE results in prevention or mitigation of epileptogenesis. The current experiments did not proof this hypothesis to be true. Furthermore, electrophysiological measurements in the DG performed to assess neuronal excitation and inhibition did not show significant differences between SSSE animals that underwent cooling and those that did not.
3.1.
Antiepileptogenic treatment strategies
The prevention of epilepsy following severe brain injuries has been one of the main focuses of epilepsy research for decades. In patients with traumatic brain injury (TBI), standard antiepileptic drugs (AED) including carbamazepine, phenobarbital, and phenytoin have been assessed in randomized controlled trials regarding their antiepileptogenic properties. All drugs failed to demonstrate any effect as compared with placebo (Temkin, 2009). In a recent phase II study, newer AEDs such as levetiracetam have been tested in patients with TBI demonstrating safety and feasibility (Klein et al., 2012). Efficacy data of this compound may be assessed in a future phase III study. Animal models of epileptogenesis
Fig. 4 – Dentate gyrus inhibition and excitation. Dentate gyrus (DG) inhibition and excitation in animals with hypothermia (black column) and with normothermia (gray column) 3 h after termination of self-sustaining status epilepticus (SSSE) or 3 h after mere administration of intraperitoneal pentobarbital (white column). (A) Extent of inhibition is indicated by paired-pulse ratio (PPR). The lower the PPR is, the stronger the DG inhibition is. At an interpulse interval of 25 ms, omnibus Kruskal–Wallis test indicates significant differences between the three groups (p¼ 0.001). Post-hoc Mann–Whitney-U test shows that PPR after cooling animals to 25 1C trends to be significantly lower (0.1670.21) compared with animals that were kept constantly at 37 1C (0.5470.21; p ¼0.027), but difference is not significant after testing for multiple comparisons. PPR was significantly higher in the normothermic group compared with electrode controls (p¼ 0.003). In contrast, there was no such difference comparing hypothermic animals with electrode controls that were not electrically stimulated and thus did not develop SSSE (p¼0.083). (B) Extent of excitation is indicated by latency between stimulus artefact and the negative peak of the excitatory post-synaptic potential. The longer the latency, the smaller is DG excitation. Omnibus Kruskal– Wallis test indicates significant differences between the three groups (p ¼0.003). Post-hoc Mann–Whitney-U test shows that hypothermic animals trended to longer latencies (8.2972.45 ms) compared with the normothermic group (4.8270.66 ms; p ¼0.014). Animals in the hypothermic group had significantly longer latencies compared with electrode controls (p¼ 0.003), while latency in the normothermic group did not differ from electrode controls (p¼0.09). Pre, before SSSE; npo0.006.
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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following fluid percussion induced brain injury or chemically or electrically induced SSSE as well tested multiple pharmacological compounds (Holtkamp and Meierkord, 2007; Loscher and Brandt, 2010) including new AED such as lacosamide (Licko et al., 2013) but prevention or mitigation of epilepsy has not been demonstrated. The antiepileptogenic inefficacy of drugs that are successful in seizure suppression has prompted the discussion that mechanisms and molecular targets underlying epileptogenesis seem to differ from those of ictogenesis (Holtkamp and Meierkord, 2007; Kobow et al., 2012).
3.2.
Hypothermia in antiepileptogenesis
Against the background of failed pharmacological approaches in prevention of epileptogenesis, the search for novel antiepileptogenic treatment strategies seems to be necessary. Thus the antiepileptogenic properties of hypothermia following experimental SSSE or traumatic brain injury have been assessed in the current study and two prior ones. Atkins et al. lowered body temperature to 33 1C for 4 h starting 30 min after application of a moderated fluid percussion pulse over the right parietal cortex (2010). Twelve weeks later, seizure susceptibility was challenged by administration of the chemoconvulsant agent pentylenetetrazole. Hypothermia resulted in a significantly lower number of seizures compared to normothermia (Atkins et al., 2010). D’Ambrosio and colleagues induced circumscribed brain trauma in rostral parasagittal brain structures by use of fluid percussion that results in epilepsy with high-frequency spontaneous recurrent seizures (2013). Three days after trauma, cooling by 2 1C was initiated with passive heat dissipation via a headset placed above the neocortical lesion. During cooling for 5 weeks but also more than 10 weeks after the end of cooling, most animals were seizure free, and in those with persisting seizures, duration was significantly shorter (D’Ambrosio et al., 2013) compared with normothermic animals. These data indicate strong antiepileptogenic properties of hypothermia. We induced cooling to 25 1C body temperature directly after termination of SSSE by i.p. pentobarbital and maintained hypothermia for a total of 3 h. As animals were sedated and thus not moving, target temperature was easily achieved. In the normothermic SSSE control group, body temperature would decrease spontaneously with pentobarbital sedation, therefore temperature of 37 1C was kept constantly by a heating lamp. Using this design and in contrast to the two previous experimental studies (Atkins et al., 2010; D’Ambrosio et al., 2013), almost all SSSE animals developed spontaneous recurrent seizures in the following 8 weeks regardless if they underwent hypothermia or not. This discrepancy may be explained by use of different animal models – fluid percussion vs. SSSE – that are likely characterized by varying inherent epileptogenicity. In the current hypothermic animals, a trend towards less severe seizures 8 weeks after SSSE was not shown to be significant following correction for multiple comparisons with measurements performed at eight different time points. One may speculate that the difference in seizure severity
would have been significant if larger numbers of animals had been examined.
3.3.
Mechanisms of action
The neurophysiological processes that are involved in antiepileptic and possible antiepileptogenic effects of hypothermia are still elusive, but a number of hypotheses have recently been summarized (Motamedi et al., 2013). General mechanisms include alteration of postsynaptic voltage-gated channels (Schiff and Somjen, 1985; Shen and Schwartzkroin, 1988; Thompson et al., 1985), disturbances of membrane properties and ion pumps (Aihara et al., 2001; Volgushev et al., 2000), and presynaptic mechanisms with marked reduction of excitatory transmitter release (Volgushev et al., 2004). Interestingly, microdialysis studies demonstrated that hypothermia results in decreased extracellular concentration (Winfree et al., 1996) or release (Van Hemelrijck et al., 2003) of the excitatory neurotransmitter glutamate. This finding is further strengthened by a model of receptor activation in hippocampal excitatory synapses demonstrating the temperature dependency of glutamate diffusion in the synaptic cleft and the dramatically reduced affinity and sensitivity of both glutamate and NMDA receptors at 25 1C (Boucher et al., 2010). Also, at least in temporo-mesial structures, evidence intensifies that NMDA receptor mediated modifications of neuronal circuits and networks majorly contribute to the pathophysiological processes underlying epileptogenesis (McNamara et al., 2006). Reduced NMDA receptor functionality would explain mitigation of epileptogenesis after application of hypothermia. This hypothesis is supported by morphological and electrophysiological findings. Mossy fiber sprouting is a common feature in hippocampal epileptogenesis (Houser et al., 1990) which is attenuated by NMDA receptor blockade (Sutula et al., 1996). Atkins et al. have demonstrated that hypothermia following fluid percussion TBI mitigates mossy fiber sprouting but its relevance in epileptogenesis is undetermined (Atkins et al., 2010). NMDA receptor blockers also reverse the early loss of GABAergic inhibition in the DG following status epilepticus (Mazarati and Wasterlain, 1997). Therefore, the antiglutamatergic effect of hypothermia would have been an explanation for potential prevention of GABAergic erosion directly after SSSE. Attenuated temporary break-down of DG inhibition directly after SSSE may contribute to a potential antiepileptogenic effect of early hypothermia. Thus reduced DG excitation and diminished loss of inhibition would point to some mechanisms of cooling that interfere with early basic processes of epileptogenesis. However, the current data shows that cooling itself is not sufficient to prevent or mitigate epileptogenesis. This finding may support the hypothesis that several interdependent factors play a role in this process (Trinka and Brigo, 2014 Epilepsia).
3.4.
Conclusion
In the current rodent model of epileptogenesis following electrically induced status epilepticus, we were not able to demonstrate that hypothermia exhibits antiepileptogenic
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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effects. All animals that underwent cooling developed epilepsy within 8 weeks of SSSE. After testing for multiple comparisons, the trend in hypothermic animals towards less severe seizures 8 weeks after SSSE did not proof to be significant. Direct comparisons of excitatory and inhibitory electrophysiological parameters in animals that underwent hypothermia and those that did not reveal significant differences. Larger number of animals may challenge the current findings and may point to some antiepileptogenic effects of post SSSE hypothermia.
4.
Experimental procedure
4.1.
Experimental animals
Male Wistar rats aged 10–12 weeks weighing 300–400 g at the time of SSSE were used in this study. Animals were housed under a 12-h-light-dark cycle, food and water were available ad libitum. All animals were implanted intracerebral electrodes for stimulation of the perforant path and recording from the DG as well as an epidural temperature probe. A total of three groups were studied at different time points for up to 8 weeks regarding occurrence and severity of spontaneous motor seizures and regarding DG excitation and inhibition. After termination of electrically induced SSSE with i.p. administered pentobarbital, one group of animals underwent hypothermia (25 1C) for 3 h, while in a control group, animals were kept at 37 1C (normothermic control). In the third group, animals were implanted intracerebral electrodes but neither SSSE nor hypothermia was induced (electrode control). To keep other variables constant, barbiturates were applied 8–10 days after electrode implantation in this latter group as well. This design was chosen to isolate the effects of electrode implantation and barbiturates from those of status epilepticus. Animals of all three groups were monitored by a videocamera system for epochs of 48 h to detect spontaneous seizures at four different time points following SSSE or other interventions: 7–9, 13–15, 26–30 and 54–58 days, in the following referred to as 1, 2, 4 and 8 weeks after SSSE (continuous monitoring 24 h/7 d not possible due to technical limitations). Electrophysiological recordings from the DG were carried out 3 h, 24 h, 4 d, 6 d, 8 d as well as 2, 4 and 8 weeks after the end of SSSE in order to assess GABAergic inhibition and excitatory parameters. To obtain intragroup control data, electrophysiological measurements were also performed directly prior to electrical stimulation. Not all animals were assessed by video and electrophysiologically at all time points. The animal experiments were carried out in accordance with the EU Directive 2010/63/EU. They were conducted in accordance with the German Animal Protection Act and were approved by the regional authority.
4.2.
Electrode implantation
Procedures have been described previously (Schmitt et al., 2005). Briefly, rats were implanted intracerebral electrodes and an epidural temperature probe following stereotactical coordinates under deep anesthesia with 52 mg/kg i.p.
7
pentobarbital (Synopharm, Barsbüttel, Germany). A bipolar stimulation electrode was implanted into the right perforant path 6.9 mm posterior and 4.1 mm lateral of bregma, a monopolar recording electrode was placed into the granule cell layer of the ipsilateral DG 3.1 mm posterior and 1.9 mm lateral of bregma. Potentials were amplified and filtered (0.1-500 Hz band pass) via a NeuroLog amplifier (Digitimer, Welwyn Garden City, UK) onto an oscilloscope and via an analogue to digital interface onto a computer using WinTIDA (HEKA Electronic, Lambrecht, Germany; sampling rate 20 kHz). The depth of electrodes was adjusted following the maximal population spike after single electrical test stimuli (150-μs monopolar pulses at 5 to 8 mA). The temperature probe consisted of a cylindrically formed thermistor (Delta, Munich, Germany) and was implanted contralaterally to the electrodes in the epidural space after skull trepanation. Electrodes and temperature probe were fit into a plastic socket and attached to the skull with dental acrylic. At the end of the procedure, animals were treated with buprenorphine (0.05 mg/kg bodyweight; Temgesic s, Essex Pharma GmbH, Germany) to relief post-surgery pain. The wound margins were adhered to the dental acrylic and healed by granulation without complication.
4.3.
Self-sustaining status epilepticus
After a post-operative recovery period of 8–10 d, the perforant path of the freely moving animals was stimulated electrically for 2 h at 20 Hz with 3–5 mA, 50- to 150-μs monopolar pulses. Along the lines described by Walker et al., we applied the following electrographical criteria to define if status had developed in the wake of stimulation: presence of spontaneous, high amplitude discharges at a frequency of Z1 Hz during and after the end of electrical stimulation (Walker et al., 1999). Three hours after the end of stimulation, SSSE was terminated by i.p. application of 30 mg/kg pentobarbital. Electrode control animals were injected the same dosage of pentobarbital without further treatment 8 d after implantation.
4.4.
Hypothermia
For induction of hypothermia, the pentobarbital-treated nonmoving animals were placed on a cool pack, frostbite damage was prevented by enveloping vulnerable body parts (ears, paws, and tail) and covering of the cool packs. When the epidural target temperature of 25 1C was reached, further decrease of temperature was prevented by a relay controlled heating lamp (mechanical relay adapter, Almemo, Holzkirchen, Germany). This system was also used to keep normothermic and electrode control animals at 37 1C (Schmitt et al., 2006).
4.5.
Seizure detection
For video-camera monitoring, the animals were placed in single plexiglass boxes with a ground of 30 40 cm and a height of 55 cm, the boxes were open at the top. Up to four animals were filmed simultaneously from above by a black and white-camera with high spectral and photo sensitivity (Kappa, Gleichen, Germany) allowing monitoring with a 12-h-light-dark-cycle. Video-data were digitalized via a video capture card (Lupustec
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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LE214, Lupus Electronics, Germany) and collected for 48 h in each episode. We employed the classification as suggested by Racine (Racine, 1972); only motor seizures with forelimb clonus (stage 3), rearing (stage 4), and rearing and falling (stage 5) were chosen for further analysis, since these seizures are reproducibly identifiable. Frequency, duration and severity of these seizures were assessed. In 32 out of 70 epochs with 48-h-video recording, we simultaneously recorded intracranial EEG from the DG. In 15 of these EEG recordings, at least one seizure, defined electrophysiologically as high-frequency (45/s) and high-amplitude (42 times baseline) discharges lasting 5 s or more (Pitkanen et al., 2005), was detected. EEG seizure numbers summed up to a total of 49. The video interpreter (M.S.) was blinded to EEG findings, and identified 35 Racine stage 3–5 motor seizure in the 15 epochs with EEG detected seizures. In post-hoc video re-analysis, the seizures detected only by EEG were associated with none or at most discrete behavioral changes such as attenuation of motor activity. These data indicate that the current seizure analysis by video recording has a sensitivity of 100% to detect Racine stage 3–5 motor seizures. Sensitivity to detect all seizures with and without motor signs is 71%.
4.6.
Electrophysiological examinations
To assess the extent of GABAergic inhibition in the DG, the paired-pulse paradigm was used as described previously (Holtkamp et al., 2005). The lowest current with 150-ms monopolar pulses given to the perforant path producing a population spike (PS) with maximal amplitude was used for the paired-pulse experiments in each session. Animals were included in this study with PS amplitudes of Z2 mV. The amplitude of the PS was calculated by averaging the descending and the ascending part of the PS. Measurements were performed at interpulse intervals (IPI) of 20, 25, 30, 40, 50, 100, 300, 500 and 1000 ms. The amplitude of the PS evoked by the test stimulus was related to the PS amplitude following the conditioning stimulus, this relation was expressed as pairedpulse ratio (PPR). With a PPR lower than 1 the test PS was inhibited, with a PPR higher than 1 the test PS was facilitated. The latency between stimulus artefact and the negative PS peak was analyzed to assess excitation of DG granule cells. For each animal, values of five subsequent measurements were averaged. Between each stimulus or pair of stimuli, a latency of 2 min was kept. Data were amplified and stored as described above.
4.7.
Statistical analysis
Electrophysiological parameters as collected in the animals before SSSE or other interventions served as intragroup controls for the eight time points after interventions. For comparison of intragroup data, the non-parametric Mann–Whitney-U (MWU) test was used. For intergroup comparisons at the individual time points, omnibus Kruskal–Wallis and post-hoc MWU test were used. Due to multiple comparisons, the Holm–Bonferronitest was applied. For comparisons of specific seizure characteristics that were assessed at four different time points, differences were considered significant with a po0.012 (po0.05/4). For comparisons of electrophysiological data that were assessed
at eight different time points, differences were considered significant with a po0.006 (po0.05/8). Data are given as mean7SD.
Acknowledgments This work was supported by a grant of the “Deutsche Forschungsgemeinschaft” to MH (Ho 3264/2-1). MH holds the “Friedrich-von-Bodelschwingh Endowed Professorship for Clinical and Experimental Epileptology” at the Department of Neurology at the Charité – Universitätsmedizin Berlin funded by von Bodelschwingh Foundation. ABK is supported by grants from the “Friedrich C. Luft” Clinical Scientist Pilot Program funded by Volkswagen Foundation and Charité Foundation. Sponsors did not have any influence on study design, on collection, analysis and interpretation of data, on writing of the report, and on decision to submit the article for publication.
r e f e r e n c e s
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Shen, K.F., Schwartzkroin, P.A., 1988. Effects of temperature alterations on population and cellular activities in hippocampal slices from mature and immature rabbit. Brain Res. 475, 305–316. Sutula, T., et al., 1996. NMDA receptor dependence of kindling and mossy fiber sprouting: evidence that the NMDA receptor regulates patterning of hippocampal circuits in the adult brain. J. Neurosci. 16, 7398–7406. Temkin, N.R., 2009. Preventing and treating posttraumatic seizures: the human experience. Epilepsia 50 (2), 10–13, http: //dx.doi.org/10.1111/j.1528-1167.2008.02005.x. Thompson, S.M., et al., 1985. Temperature dependence of intrinsic membrane properties and synaptic potentials in hippocampal CA1 neurons in vitro. J. Neurosci. 5, 817–824. Trinka, E., Brigo, F., 2014. Antiepileptogenesis in humans: disappointing clinical evidence and ways to move forward. Curr. Opin. Neurol. 27, 227–235, http://dx.doi.org/10.1097/ WCO.0000000000000067. Van Hemelrijck, A., et al., 2003. Effect of resuscitative mild hypothermia on glutamate and dopamine release, apoptosis and ischaemic brain damage in the endothelin-1 rat model for focal cerebral ischaemia. J. Neurochem. 87, 66–75. Volgushev, M., et al., 2004. Probability of transmitter release at neocortical synapses at different temperatures. J. Neurophysiol. 92, 212–220. Volgushev, M., et al., 2000. Membrane properties and spike generation in rat visual cortical cells during reversible cooling. J. Physiol. 522 (1), 59–76. Walker, M.C., et al., 1999. Halothane as a neuroprotectant during constant stimulation of the perforant path. Epilepsia 40, 359–364. Winfree, C.J., et al., 1996. Mild hypothermia reduces penumbral glutamate levels in the rat permanent focal cerebral ischemia model. Neurosurgery 38, 1216–1222.
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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Research Report
Hypothermia did not prevent epilepsy following experimental status epilepticus Mirja Steinbrennera,1, Alexander B. Kowskia,1, Friedhelm C. Schmittb, Martin Holtkampa,n a Epilepsy-Center Berlin-Brandenburg, Department of Neurology, Charité - Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany b Department of Neurology, Universitätsklinik Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany
art i cle i nfo
ab st rac t
Article history:
In epilepsy research, one of the major challenges is to prevent or at least mitigate
Accepted 12 May 2014
development of epilepsy following acquired brain insult by early therapeutic interventions. So far, all pharmacological antiepileptogenic treatment approaches were largely unsuccess-
Keywords:
ful in clinical trials and in experimental animal studies. In a well-established rat model
Brain insult
of chronic epilepsy following self-sustaining status epilepticus (SSSE), we assessed the
Cooling
antiepileptogenic properties of 3-h-cooling induced directly after the end of SSSE. Occur-
Excitation
rence of spontaneous seizures and seizure severity up to 8 weeks after SSSE were compared
Inhibition
with normothermic SSSE controls. Furthermore, electrophysiological parameters assessing
Spontaneous seizure
inhibition and excitation in the dentate gyrus were assessed at multiple time points. Post SSSE hypothermia did not prevent the occurrence of seizures in any animal. Eight weeks after SSSE, Racine motor seizures trended to be less severe following cooling (4.070.6) compared with normothermic controls (4.870.2) but the difference was not significant when testing for multiple comparisons. Early loss of inhibition that is typically seen following SSSE was somewhat attenuated in cooled animals 3 h after SSSE as expressed by smaller pairedpulse ratios (PPR; 0.1670.21) compared with normothermic controls (0.5470.21) but difference was not significant either. Latency between stimulus artefact and excitatory post-synaptic potential 3 h after SSSE, reciprocally reflecting neuronal excitation, was higher in animals that underwent hypothermia (8.2972.45 ms)
compared with controls
(4.8270.66 ms), difference was not significant after correction for multiple comparisons. In summary, the current experiments were not able to demonstrate prevention or mitigation of epileptogenesis with immediate short-term cooling following SSSE. & 2014 Published by Elsevier B.V.
n
Corresponding author. Fax: þ49 30 450 560 932. E-mail addresses: [email protected] (M. Steinbrenner), [email protected] (A.B. Kowski), [email protected] (F.C. Schmitt), [email protected] (M. Holtkamp). 1 MS and ABK contributed equally to this manuscript. http://dx.doi.org/10.1016/j.brainres.2014.05.018 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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1.
Introduction
Antiepileptic treatment commonly refers to suppression of unprovoked epileptic seizures in established epilepsy that in fact represents an antiictal treatment strategy. In patients with acquired brain insults such as traumatic brain injury or status epilepticus, which imply a high risk of subsequent epilepsy, early therapeutic interventions aim to completely prevent or at least mitigate the development of epilepsy representing an antiepileptogenic approach (Pitkanen and Lukasiuk, 2011). Antiepileptic drugs that are successful antiictally and a plethora of other pharmacological agents with various mechanisms of action have been assessed regarding their antiepileptogenic properties in a few clinical trials and manifoldly in animal models of epileptogenesis induced by diverse brain insults. In summary, none of these studies demonstrated an antiepileptogenic effect (Holtkamp and Meierkord, 2007; Kobow et al., 2012; Loscher and Brandt, 2010). Hypothermia, a non-pharmological treatment approach, is well-known to reduce cortical excitability. Thus, it has been applied in patients to suppress circumscribed intraoperative spiking in epilepsy surgery (Karkar et al., 2002) and to contribute to management of refractory status epilepticus (Guilliams et al., 2013; Orlowski et al., 1984). In an experimental model of electrically induced self-sustaining status epilepticus (SSSE), our group has demonstrated strong anticonvulsant effects of moderate (30 1C) and deep (20 1C) hypothermia eventually terminating continuous seizure activity (Kowski et al., 2012; Schmitt et al., 2006). In animal models of fluid percussion injury, seizure susceptibility was attenuated by early hypothermia (Atkins et al., 2010) and epileptogenesis was largely prevented by mildly lowering perilesional temperature by 2 1C for 5 weeks (D’Ambrosio et al., 2013). In the latter study, interventional hypothermia was initiated 3 d after traumatic brain injury. Relevant pathophysiological changes promoting epileptogenesis such as accumulation of N-methyl-D-aspartate (NMDA) receptors (McNamara et al., 2006) have been described to occur in the first hour after experimental status epilepticus (Naylor
et al., 2013). Due to some antiglutamatergic effects of cooling (Van Hemelrijck et al., 2003; Winfree et al., 1996), we hypothesized that induction of hypothermia for 3 h directly after termination of SSSE has antiepileptogenic properties. At various time points up to 8 weeks following SSSE, we assessed occurrence and severity of spontaneous seizures by intermittent video recording and dentate gyrus (DG) inhibition and excitation by in vivo electrophysiological measurements (for overview, see Fig. 1).
2.
Results
2.1.
Self-sustaining status epilepticus
In 23 animals, the perforant path was stimulated electrically for 2 h resulting in SSSE in 21 animals. The remainder did not fulfill our inclusion criteria as regularly occurring spontaneous discharges (Z 1 Hz) at the end of stimulation were lacking. These 21 animals maintained SSSE for the next 3 h. Two animals died directly after pentobarbital-induced termination of SSSE. Thus, a total of 19 animals were eligible for post-SSSE hypothermia or normothermia and for subsequent video recording and electrophysiological studies. In the electrode control group, nine animals were injected intraperitoneally (i.p.) pentobarbital at an index time point without prior induction of SSSE.
2.2.
Induction of hypothermia
Five minutes after i.p. administration of 30 mg/kg pentobarbital, SSSE animals either underwent cooling down to 25 1C (n ¼9) or were maintained at 37 1C core temperature (normothermic controls, n¼ 10) for 3 h. Electrode controls (n¼ 9) were kept at 37 1C for 5 h after administration of pentobarbital. In cooled animals, the target temperature was reached after 40713 min. At the end of induced hypothermia, this group of rats was rewarmed to 37 1C over another 2 h. Fig. 2 illustrates the course of body temperature in both groups of animals following SSSE.
Fig. 1 – Study overview. Animals of all three groups underwent implantation of electrodes into the right perforant path and the ipsilateral dentate gyrus. Two groups underwent electrical induction of self-sustaining status epilepticus (SSSE) that after 3 h was terminated with pentobarbital, while animals of the electrode control group remained unstimulated but were also administered pentobarbital. After the end of SSSE, one of the two groups underwent immediate cooling to 25 1C body temperature that was maintained for 3 h. The other post-SSSE group and the electrode controls were kept at 37 1C. Animals of all groups were video-monitored for periods of 48 h to detect behavioral seizures 1, 2, 4 and 8 weeks after SSSE. All animals underwent electrophysiological (e’ph) measurements to assess dentate gyrus inhibition and excitation 3 h, 24 h, 4 d, 6 d, 8 d, 2 weeks, 4 weeks and 8 weeks after SSSE. Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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2.3.
Spontaneous seizures
detected seizures without behavioral changes at weeks 2 and 8. Frequency distributions of animals with spontaneous seizures between SSSE groups were not significantly different at any time point assessed (table 1). In epileptic animals, mean number of seizures in all epochs of 48-h-video recording was 2.472.8 without any significant differences between groups at all and at the four defined time points (data not shown). Mean duration of motor seizures was 30.775.9 s in animals that underwent cooling and 32.577.1 s in normothermic controls, the difference was not significant. Motor seizure severity was assessed by the Racine scale. We calculated the mean score in each animal at each time point. Eight weeks post SSSE, animals that underwent cooling trended to lower scores (4.070.6; n¼ 6) compared with normothermic controls (4.870.2; n¼6) but difference was not significant when taking multiple comparisons into account. At all other time points assessed, we were not able to demonstrate statistically significant differences in seizure severity either (table 1).
One, 2, 4 and 8 weeks after SSSE or exclusive pentobarbital injection, animals underwent 48-h-periods of continuous video-monitoring. None of the animals in the electrode control group developed spontaneous seizures at any time point. In animals that underwent hypothermia, one rat died 2 weeks after SSSE, without any seizures detected in videorecordings in week 1 or 2. Another animal died after video recording 4 weeks post SSSE but had spontaneous seizures before. Eventually eight out of eight eligible rats were epileptic at the end of follow-up, i.e. they exhibited spontaneous seizures at least at one time point assessed. In normothermic SSSE animals, two died during 8-week-follow-up, both had seizures before. At the end of the study, nine out of 10 animals were epileptic. In the single normothermic rat without Racine stage 3 to 5 motor seizures, EEG monitoring
2.4.
Electrophysiological findings
2.4.1.
GABAergic inhibition
Prior to SSSE, small paired-pulse ratios (PPR) at short interpulse intervals (IPI) of 20 ms (0.0370.12), 25 ms (0.0370.08) and 30 ms (0.0770.14) in animals of all three groups indicated strong physiological inhibition in the DG. Typically, an IPI of 100 ms resulted in facilitation and IPIs between 300 and 1000 ms in weak inhibition. At any IPI, there was no significant difference between all three groups (data not shown). In the electrode control group, PPR as compared to baseline (the data collected before the intervention) was not significantly altered at the majority of time points assessed after administration of pentobarbital. Only at the time point 3 h after SSSE and at an IPI of 50 ms, PPR was significantly decreased as compared with pre-SSSE baseline (p¼ 0.001). As pentobarbital was administered in electrode controls and in the two post-SSSE groups of animals, possible electrophysiological differences in hypothermic animals compared with other groups can be attributed to cooling. In the 8 weeks following SSSE, in normothermic animals the changes of PPR at IPIs of 20–30 ms indicate an initial relevant loss of inhibition with a maximum 24 h after the end of status epilepticus. Impaired inhibition sustained for the
Fig. 2 – Course of body temperature. Body core temperature of experimental animals measured epidurally. Hours 0–2 indicate electrical stimulation of the dentate gyrus, hours 2–5 indicate self-sustaining status epilepticus (SSSE). Baseline temperature is approximately 37 1C. During 2-helectrial stimulation, animals exhibit numerous motor seizures (Racine class 4 and 5, i.e. rearing and falling) resulting in increased body temperature of 38–39 1C. Animals that undergo hypothermia (grey line) after termination of SSSE, reach the target temperature of 25 1C within 40713 min. Three hours after onset of cooling, animals are rewarmed to baseline temperature. Body temperature of animals in the normothermic group (black line) is kept constantly at 37 1C by a relais-controlled heating lamp.
Table 1 – Seizure occurrence and severity. Week
1 2 4 8
Animals with seizures(n/n)
Racine score(mean7SD)
Hypothermic group
Normothermic group
p-values
0/9 1/9 5/8 6/7
0/10 2/10 5/9 6/8
n.a. 0.542 0.581 0.554
n
Hypothermic group
Normothermic group
p-valuesnn
n.a. 4.070.0 4.370.6 4.070.6
n.a. 4.370.4 4.470.5 4.870.2
n.a. 0.667 0.792 0.026
n, number; SD, standard deviation; n.a., not applicable. Fischer's exact test. nn Mann–Whitney-U test, 2-sided, following Holms-Bonferroni test to correct for multiple comparisons differences are significant with po0.012. n
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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Fig. 3 – Course of inhibition following self-sustaining status epilepticus. (A) Following self-sustaining status epilepticus (SSSE), strong inhibition as seen at baseline is dramatically impaired already 3 h after termination of status epilepticus reaching its maximum at 24 h post as indicated by significantly increased paired-pulse ratios (PPR). In the following weeks, PPRs are gradually restored, baseline values are achieved 8 weeks after SSSE. Pre, before SSSE; h, hour; d, day; w, week; * po0.006. (B) Example of excitatory post-synaptic potentials and population spikes in response to conditioning and test stimuli at an interpulse interval of 20 ms before and at eight time points after SSSE. next 4 weeks and recovers to baseline 8 weeks after SSSE (Fig. 3a). An example of field potentials in response to paired stimuli at an IPI of 20 ms in the course of 8 weeks after SSSE in a normothermic animal is given in Fig. 3b.
In animals that underwent post-SSSE hypothermia, the very early loss of GABAergic inhibition 3 h after the end of SSSE trended to be mitigated. At an IPI of 25 ms, increase of PPR was less pronounced when comparing SSSE animals with
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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cooling (0.1670.21) to those with normothermia (0.5470.21; p¼ 0.027; Fig. 4a). After testing for multiple comparisons, the difference did not reach a significant level. While normothermic SSSE animals had significantly higher PPRs than electrode controls (0.070.0; p ¼0.001) indicating relevant loss of inhibition, this difference was compensated for by hypothermia (p¼ 0.083). This indirectly suggests that cooling diminished the loss of inhibition 3 h after the end of SSSE.
2.4.2.
Excitatory parameter
Analogue to the inhibitory data, latency between the stimulus and the negative peak of the population spike prior to SSSE was not significantly different between all three groups (3.6070.47 ms in all animals). In electrode controls, latency remained unchanged within the 8 weeks following administration of pentobarbital (data not shown) excluding any impact of the sedative agent. Following SSSE, in normothermic animals latency was significantly longer at one time point after SSSE (24 h) as compared with baseline (p ¼ 0.003). Cooling to 25 1C for 3 h after SSSE resulted in a trend towards longer latency comparing SSSE animals with cooling (8.2972.45 ms) to those with normothermia (4.8270.66 ms; p¼ 0.014) (Fig. 4b). This would indicate reduced neuronal excitation, but the difference was not significant when testing for multiple comparisons. While latency in normotermic SSSE animals did not differ from electrode controls (4.0570.51), latency in hypothermic SSSE animals was significant longer compared with electrode controls (p ¼0.003). This indirectly suggests that 3 h after the end of SSSE cooling results in prolonged latency and thus decreased neuronal excitation.
3.
5
Discussion
We tested the hypothesis if hypothermia applied to rats f or 3 h directly following termination of SSSE results in prevention or mitigation of epileptogenesis. The current experiments did not proof this hypothesis to be true. Furthermore, electrophysiological measurements in the DG performed to assess neuronal excitation and inhibition did not show significant differences between SSSE animals that underwent cooling and those that did not.
3.1.
Antiepileptogenic treatment strategies
The prevention of epilepsy following severe brain injuries has been one of the main focuses of epilepsy research for decades. In patients with traumatic brain injury (TBI), standard antiepileptic drugs (AED) including carbamazepine, phenobarbital, and phenytoin have been assessed in randomized controlled trials regarding their antiepileptogenic properties. All drugs failed to demonstrate any effect as compared with placebo (Temkin, 2009). In a recent phase II study, newer AEDs such as levetiracetam have been tested in patients with TBI demonstrating safety and feasibility (Klein et al., 2012). Efficacy data of this compound may be assessed in a future phase III study. Animal models of epileptogenesis
Fig. 4 – Dentate gyrus inhibition and excitation. Dentate gyrus (DG) inhibition and excitation in animals with hypothermia (black column) and with normothermia (gray column) 3 h after termination of self-sustaining status epilepticus (SSSE) or 3 h after mere administration of intraperitoneal pentobarbital (white column). (A) Extent of inhibition is indicated by paired-pulse ratio (PPR). The lower the PPR is, the stronger the DG inhibition is. At an interpulse interval of 25 ms, omnibus Kruskal–Wallis test indicates significant differences between the three groups (p¼ 0.001). Post-hoc Mann–Whitney-U test shows that PPR after cooling animals to 25 1C trends to be significantly lower (0.1670.21) compared with animals that were kept constantly at 37 1C (0.5470.21; p ¼0.027), but difference is not significant after testing for multiple comparisons. PPR was significantly higher in the normothermic group compared with electrode controls (p¼ 0.003). In contrast, there was no such difference comparing hypothermic animals with electrode controls that were not electrically stimulated and thus did not develop SSSE (p¼0.083). (B) Extent of excitation is indicated by latency between stimulus artefact and the negative peak of the excitatory post-synaptic potential. The longer the latency, the smaller is DG excitation. Omnibus Kruskal– Wallis test indicates significant differences between the three groups (p ¼0.003). Post-hoc Mann–Whitney-U test shows that hypothermic animals trended to longer latencies (8.2972.45 ms) compared with the normothermic group (4.8270.66 ms; p ¼0.014). Animals in the hypothermic group had significantly longer latencies compared with electrode controls (p¼ 0.003), while latency in the normothermic group did not differ from electrode controls (p¼0.09). Pre, before SSSE; npo0.006.
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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following fluid percussion induced brain injury or chemically or electrically induced SSSE as well tested multiple pharmacological compounds (Holtkamp and Meierkord, 2007; Loscher and Brandt, 2010) including new AED such as lacosamide (Licko et al., 2013) but prevention or mitigation of epilepsy has not been demonstrated. The antiepileptogenic inefficacy of drugs that are successful in seizure suppression has prompted the discussion that mechanisms and molecular targets underlying epileptogenesis seem to differ from those of ictogenesis (Holtkamp and Meierkord, 2007; Kobow et al., 2012).
3.2.
Hypothermia in antiepileptogenesis
Against the background of failed pharmacological approaches in prevention of epileptogenesis, the search for novel antiepileptogenic treatment strategies seems to be necessary. Thus the antiepileptogenic properties of hypothermia following experimental SSSE or traumatic brain injury have been assessed in the current study and two prior ones. Atkins et al. lowered body temperature to 33 1C for 4 h starting 30 min after application of a moderated fluid percussion pulse over the right parietal cortex (2010). Twelve weeks later, seizure susceptibility was challenged by administration of the chemoconvulsant agent pentylenetetrazole. Hypothermia resulted in a significantly lower number of seizures compared to normothermia (Atkins et al., 2010). D’Ambrosio and colleagues induced circumscribed brain trauma in rostral parasagittal brain structures by use of fluid percussion that results in epilepsy with high-frequency spontaneous recurrent seizures (2013). Three days after trauma, cooling by 2 1C was initiated with passive heat dissipation via a headset placed above the neocortical lesion. During cooling for 5 weeks but also more than 10 weeks after the end of cooling, most animals were seizure free, and in those with persisting seizures, duration was significantly shorter (D’Ambrosio et al., 2013) compared with normothermic animals. These data indicate strong antiepileptogenic properties of hypothermia. We induced cooling to 25 1C body temperature directly after termination of SSSE by i.p. pentobarbital and maintained hypothermia for a total of 3 h. As animals were sedated and thus not moving, target temperature was easily achieved. In the normothermic SSSE control group, body temperature would decrease spontaneously with pentobarbital sedation, therefore temperature of 37 1C was kept constantly by a heating lamp. Using this design and in contrast to the two previous experimental studies (Atkins et al., 2010; D’Ambrosio et al., 2013), almost all SSSE animals developed spontaneous recurrent seizures in the following 8 weeks regardless if they underwent hypothermia or not. This discrepancy may be explained by use of different animal models – fluid percussion vs. SSSE – that are likely characterized by varying inherent epileptogenicity. In the current hypothermic animals, a trend towards less severe seizures 8 weeks after SSSE was not shown to be significant following correction for multiple comparisons with measurements performed at eight different time points. One may speculate that the difference in seizure severity
would have been significant if larger numbers of animals had been examined.
3.3.
Mechanisms of action
The neurophysiological processes that are involved in antiepileptic and possible antiepileptogenic effects of hypothermia are still elusive, but a number of hypotheses have recently been summarized (Motamedi et al., 2013). General mechanisms include alteration of postsynaptic voltage-gated channels (Schiff and Somjen, 1985; Shen and Schwartzkroin, 1988; Thompson et al., 1985), disturbances of membrane properties and ion pumps (Aihara et al., 2001; Volgushev et al., 2000), and presynaptic mechanisms with marked reduction of excitatory transmitter release (Volgushev et al., 2004). Interestingly, microdialysis studies demonstrated that hypothermia results in decreased extracellular concentration (Winfree et al., 1996) or release (Van Hemelrijck et al., 2003) of the excitatory neurotransmitter glutamate. This finding is further strengthened by a model of receptor activation in hippocampal excitatory synapses demonstrating the temperature dependency of glutamate diffusion in the synaptic cleft and the dramatically reduced affinity and sensitivity of both glutamate and NMDA receptors at 25 1C (Boucher et al., 2010). Also, at least in temporo-mesial structures, evidence intensifies that NMDA receptor mediated modifications of neuronal circuits and networks majorly contribute to the pathophysiological processes underlying epileptogenesis (McNamara et al., 2006). Reduced NMDA receptor functionality would explain mitigation of epileptogenesis after application of hypothermia. This hypothesis is supported by morphological and electrophysiological findings. Mossy fiber sprouting is a common feature in hippocampal epileptogenesis (Houser et al., 1990) which is attenuated by NMDA receptor blockade (Sutula et al., 1996). Atkins et al. have demonstrated that hypothermia following fluid percussion TBI mitigates mossy fiber sprouting but its relevance in epileptogenesis is undetermined (Atkins et al., 2010). NMDA receptor blockers also reverse the early loss of GABAergic inhibition in the DG following status epilepticus (Mazarati and Wasterlain, 1997). Therefore, the antiglutamatergic effect of hypothermia would have been an explanation for potential prevention of GABAergic erosion directly after SSSE. Attenuated temporary break-down of DG inhibition directly after SSSE may contribute to a potential antiepileptogenic effect of early hypothermia. Thus reduced DG excitation and diminished loss of inhibition would point to some mechanisms of cooling that interfere with early basic processes of epileptogenesis. However, the current data shows that cooling itself is not sufficient to prevent or mitigate epileptogenesis. This finding may support the hypothesis that several interdependent factors play a role in this process (Trinka and Brigo, 2014 Epilepsia).
3.4.
Conclusion
In the current rodent model of epileptogenesis following electrically induced status epilepticus, we were not able to demonstrate that hypothermia exhibits antiepileptogenic
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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effects. All animals that underwent cooling developed epilepsy within 8 weeks of SSSE. After testing for multiple comparisons, the trend in hypothermic animals towards less severe seizures 8 weeks after SSSE did not proof to be significant. Direct comparisons of excitatory and inhibitory electrophysiological parameters in animals that underwent hypothermia and those that did not reveal significant differences. Larger number of animals may challenge the current findings and may point to some antiepileptogenic effects of post SSSE hypothermia.
4.
Experimental procedure
4.1.
Experimental animals
Male Wistar rats aged 10–12 weeks weighing 300–400 g at the time of SSSE were used in this study. Animals were housed under a 12-h-light-dark cycle, food and water were available ad libitum. All animals were implanted intracerebral electrodes for stimulation of the perforant path and recording from the DG as well as an epidural temperature probe. A total of three groups were studied at different time points for up to 8 weeks regarding occurrence and severity of spontaneous motor seizures and regarding DG excitation and inhibition. After termination of electrically induced SSSE with i.p. administered pentobarbital, one group of animals underwent hypothermia (25 1C) for 3 h, while in a control group, animals were kept at 37 1C (normothermic control). In the third group, animals were implanted intracerebral electrodes but neither SSSE nor hypothermia was induced (electrode control). To keep other variables constant, barbiturates were applied 8–10 days after electrode implantation in this latter group as well. This design was chosen to isolate the effects of electrode implantation and barbiturates from those of status epilepticus. Animals of all three groups were monitored by a videocamera system for epochs of 48 h to detect spontaneous seizures at four different time points following SSSE or other interventions: 7–9, 13–15, 26–30 and 54–58 days, in the following referred to as 1, 2, 4 and 8 weeks after SSSE (continuous monitoring 24 h/7 d not possible due to technical limitations). Electrophysiological recordings from the DG were carried out 3 h, 24 h, 4 d, 6 d, 8 d as well as 2, 4 and 8 weeks after the end of SSSE in order to assess GABAergic inhibition and excitatory parameters. To obtain intragroup control data, electrophysiological measurements were also performed directly prior to electrical stimulation. Not all animals were assessed by video and electrophysiologically at all time points. The animal experiments were carried out in accordance with the EU Directive 2010/63/EU. They were conducted in accordance with the German Animal Protection Act and were approved by the regional authority.
4.2.
Electrode implantation
Procedures have been described previously (Schmitt et al., 2005). Briefly, rats were implanted intracerebral electrodes and an epidural temperature probe following stereotactical coordinates under deep anesthesia with 52 mg/kg i.p.
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pentobarbital (Synopharm, Barsbüttel, Germany). A bipolar stimulation electrode was implanted into the right perforant path 6.9 mm posterior and 4.1 mm lateral of bregma, a monopolar recording electrode was placed into the granule cell layer of the ipsilateral DG 3.1 mm posterior and 1.9 mm lateral of bregma. Potentials were amplified and filtered (0.1-500 Hz band pass) via a NeuroLog amplifier (Digitimer, Welwyn Garden City, UK) onto an oscilloscope and via an analogue to digital interface onto a computer using WinTIDA (HEKA Electronic, Lambrecht, Germany; sampling rate 20 kHz). The depth of electrodes was adjusted following the maximal population spike after single electrical test stimuli (150-μs monopolar pulses at 5 to 8 mA). The temperature probe consisted of a cylindrically formed thermistor (Delta, Munich, Germany) and was implanted contralaterally to the electrodes in the epidural space after skull trepanation. Electrodes and temperature probe were fit into a plastic socket and attached to the skull with dental acrylic. At the end of the procedure, animals were treated with buprenorphine (0.05 mg/kg bodyweight; Temgesic s, Essex Pharma GmbH, Germany) to relief post-surgery pain. The wound margins were adhered to the dental acrylic and healed by granulation without complication.
4.3.
Self-sustaining status epilepticus
After a post-operative recovery period of 8–10 d, the perforant path of the freely moving animals was stimulated electrically for 2 h at 20 Hz with 3–5 mA, 50- to 150-μs monopolar pulses. Along the lines described by Walker et al., we applied the following electrographical criteria to define if status had developed in the wake of stimulation: presence of spontaneous, high amplitude discharges at a frequency of Z1 Hz during and after the end of electrical stimulation (Walker et al., 1999). Three hours after the end of stimulation, SSSE was terminated by i.p. application of 30 mg/kg pentobarbital. Electrode control animals were injected the same dosage of pentobarbital without further treatment 8 d after implantation.
4.4.
Hypothermia
For induction of hypothermia, the pentobarbital-treated nonmoving animals were placed on a cool pack, frostbite damage was prevented by enveloping vulnerable body parts (ears, paws, and tail) and covering of the cool packs. When the epidural target temperature of 25 1C was reached, further decrease of temperature was prevented by a relay controlled heating lamp (mechanical relay adapter, Almemo, Holzkirchen, Germany). This system was also used to keep normothermic and electrode control animals at 37 1C (Schmitt et al., 2006).
4.5.
Seizure detection
For video-camera monitoring, the animals were placed in single plexiglass boxes with a ground of 30 40 cm and a height of 55 cm, the boxes were open at the top. Up to four animals were filmed simultaneously from above by a black and white-camera with high spectral and photo sensitivity (Kappa, Gleichen, Germany) allowing monitoring with a 12-h-light-dark-cycle. Video-data were digitalized via a video capture card (Lupustec
Please cite this article as: Steinbrenner, M., et al., Hypothermia did not prevent epilepsy following experimental status epilepticus. Brain Research (2014), http://dx.doi.org/10.1016/j.brainres.2014.05.018
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LE214, Lupus Electronics, Germany) and collected for 48 h in each episode. We employed the classification as suggested by Racine (Racine, 1972); only motor seizures with forelimb clonus (stage 3), rearing (stage 4), and rearing and falling (stage 5) were chosen for further analysis, since these seizures are reproducibly identifiable. Frequency, duration and severity of these seizures were assessed. In 32 out of 70 epochs with 48-h-video recording, we simultaneously recorded intracranial EEG from the DG. In 15 of these EEG recordings, at least one seizure, defined electrophysiologically as high-frequency (45/s) and high-amplitude (42 times baseline) discharges lasting 5 s or more (Pitkanen et al., 2005), was detected. EEG seizure numbers summed up to a total of 49. The video interpreter (M.S.) was blinded to EEG findings, and identified 35 Racine stage 3–5 motor seizure in the 15 epochs with EEG detected seizures. In post-hoc video re-analysis, the seizures detected only by EEG were associated with none or at most discrete behavioral changes such as attenuation of motor activity. These data indicate that the current seizure analysis by video recording has a sensitivity of 100% to detect Racine stage 3–5 motor seizures. Sensitivity to detect all seizures with and without motor signs is 71%.
4.6.
Electrophysiological examinations
To assess the extent of GABAergic inhibition in the DG, the paired-pulse paradigm was used as described previously (Holtkamp et al., 2005). The lowest current with 150-ms monopolar pulses given to the perforant path producing a population spike (PS) with maximal amplitude was used for the paired-pulse experiments in each session. Animals were included in this study with PS amplitudes of Z2 mV. The amplitude of the PS was calculated by averaging the descending and the ascending part of the PS. Measurements were performed at interpulse intervals (IPI) of 20, 25, 30, 40, 50, 100, 300, 500 and 1000 ms. The amplitude of the PS evoked by the test stimulus was related to the PS amplitude following the conditioning stimulus, this relation was expressed as pairedpulse ratio (PPR). With a PPR lower than 1 the test PS was inhibited, with a PPR higher than 1 the test PS was facilitated. The latency between stimulus artefact and the negative PS peak was analyzed to assess excitation of DG granule cells. For each animal, values of five subsequent measurements were averaged. Between each stimulus or pair of stimuli, a latency of 2 min was kept. Data were amplified and stored as described above.
4.7.
Statistical analysis
Electrophysiological parameters as collected in the animals before SSSE or other interventions served as intragroup controls for the eight time points after interventions. For comparison of intragroup data, the non-parametric Mann–Whitney-U (MWU) test was used. For intergroup comparisons at the individual time points, omnibus Kruskal–Wallis and post-hoc MWU test were used. Due to multiple comparisons, the Holm–Bonferronitest was applied. For comparisons of specific seizure characteristics that were assessed at four different time points, differences were considered significant with a po0.012 (po0.05/4). For comparisons of electrophysiological data that were assessed
at eight different time points, differences were considered significant with a po0.006 (po0.05/8). Data are given as mean7SD.
Acknowledgments This work was supported by a grant of the “Deutsche Forschungsgemeinschaft” to MH (Ho 3264/2-1). MH holds the “Friedrich-von-Bodelschwingh Endowed Professorship for Clinical and Experimental Epileptology” at the Department of Neurology at the Charité – Universitätsmedizin Berlin funded by von Bodelschwingh Foundation. ABK is supported by grants from the “Friedrich C. Luft” Clinical Scientist Pilot Program funded by Volkswagen Foundation and Charité Foundation. Sponsors did not have any influence on study design, on collection, analysis and interpretation of data, on writing of the report, and on decision to submit the article for publication.
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