Epilepsy Research (2014) 108, 802—805
journal homepage: www.elsevier.com/locate/epilepsyres
SHORT COMMUNICATION
Prolonged depth electrode implantation in the limbic system increases the severity of status epilepticus in rats Jens P. Bankstahl a,b,1, Claudia Brandt a,b, Wolfgang Löscher a,b,∗ a
Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany b Center for Systems Neuroscience, Hannover, Germany Received 28 August 2013; received in revised form 23 January 2014; accepted 29 January 2014 Available online 8 February 2014
KEYWORDS Epilepsy; Kindling; Seizures; Amygdala; Blood—brain barrier; Hemorrhage
Summary Chronically implanted intracranial depth electrodes are widely used for studying electroencephalographic activities in deep cerebral locations and for electrical stimulation of such locations. We have previously reported that prolonged implantation of an electrode in the basolateral amygdala (BLA) of rats facilitates subsequent kindling from this site, indicating a pro-kindling or pro-epileptogenic effect. To further characterize this phenomenon, we analyzed data from experiments in which we induced a self-sustaining status epilepticus (SSSE) by BLA stimulation following different periods of post-surgical delay. In a total of 183 Sprague-Dawley rats, three groups with different periods of postsurgical delay to onset of electrical stimulation were compared: group 1 (16 days on average), group 2 (28 days) and group 3 (48 days). Three types of SSSE were observed after BLA stimulation: type 1 (nonconvulsive), type 2 (nonconvulsive occasionally interrupted by generalized convulsive activity), and type 3 (generalized convulsive). While groups 1 and 2 did not differ in the frequency of these SSSE types, the group with the longest interval between electrode implantation and stimulation (group 3) showed significantly more severe SSSE than the two other groups. The data indicate that intracranial electrode implantation may increase the sensitivity of the implanted area to seizure induction, extending previous findings in the kindling model. Potential mechanisms of these findings include the functional consequences of local microhemorrhages and blood—brain barrier destruction. © 2014 Elsevier B.V. All rights reserved.
Abbreviations: ADT, afterdischarge threshold; BLA, basolateral amygdala; SE, status epilepticus; SRS, spontaneous recurrent seizures; SSSE, self-sustaning status epilepticus. ∗ Corresponding author at: Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Bünteweg 17, D30559 Hannover, Germany. Tel.: +49 511 856 8721; fax: +49 511 953 8581. E-mail address: [email protected] (W. Löscher). 1 Present address: Preclinical Molecular Imaging, Department of Nuclear Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. 0920-1211/$ — see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2014.01.025
Intracranial electrodes increase seizure susceptibility
Introduction It is generally assumed that the use of intracranial recording with depth electrodes in the presurgical evaluation of patients with medically intractable epilepsy is a reasonably safe method (Van Loo et al., 2011). Similarly, in experimental studies with chronic implantation of depth electrodes into specific brain regions for recording of electroencephalographic activity or electrical stimulation of discrete regions, it is often implicitly assumed that the implantation of electrodes has little if any effect on brain function and related behaviors. However, we and others have previously shown in rats that chronic implantation of electrodes constructed from a variety of materials into different parts of the brain may induce lasting neurochemical, neurophysiological, morphological, and behavioral alterations, including a prokindling effect (Blackwood et al., 1982; Löscher et al., 1993, 1995, 1999; Niespodziany et al., 1999). Boast et al. (1976) demonstrated in rodents that electrode implantation per se may result in functionally significant hemorrhagic vascular damage and consequent release of blood into brain tissue, leading to iron deposits, which may induce chronic focal epileptic discharges (Willmore et al., 1978). In epilepsy patients, the incidence of intracranial hemorrhage associated with depth electrodes is up to 14% (Van Loo et al., 2011). Furthermore, electrode implantation locally destroys the blood—brain barrier, which may lead to extravasation of albumin and, hence, development of an epileptic focus (Friedman et al., 2009). From these data, we recently hypothesized that kindling, i.e., a model in which repeated administration of initially subconvulsive electrical stimuli via a depth electrode in the limbic system induces seizures that progressively increase in severity and duration, may represent a model in which the consequences of traumatic brain injury (by electrode implantation) are facilitated by electrical stimulation (Löscher and Brandt, 2010). Over recent years, models in which a status epilepticus (SE) induced by electrical or chemical stimuli results in the development of spontaneous recurrent seizures (SRS) have progressively replaced kindling as a model of epileptogenesis (Löscher and Brandt, 2010). We have used such a model, in which a self-sustaining SE (SSSE) is induced by electrical stimulation of the anterior basolateral amygdala (BLA; Brandt et al., 2003), extensively over recent years and had repeatedly the impression that a long interval (≥4 weeks) between electrode implantation and SE induction increases the incidence and severity of SSSE. We therefore performed a retrospective analysis of data to determine whether prolonged electrode implantation in the limbic system alters the sensitivity of rats to SE induction.
Methods As in previous studies of our group on the SE model used in this study (Brandt et al., 2003), adult female SpragueDawley rats (body weight 200—230 g) were obtained from Harlan-Winkelmann (Borchen, Germany) or Harlan (Horst, Netherlands) and adapted for at least one week before onset of the experiments. Maintenance of rats was as previously described (Brandt et al., 2003). All experiments were
803 done in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize pain or discomfort of the animals used. At time of electrode implantation, all rats were about 80 days of age. For electrode implantation, rats were anesthetized with chloral hydrate (360 mg/kg i.p.). A bipolar electrode was stereotaxically implanted into the right BLA and served as stimulation- and recording-electrode. As in recent studies of our group, the electrode consisted of two twisted Teflon-coated, 0.2 mm diameter, stainless steel wires separated by 0.5 mm at the tip. The stereotaxic coordinates in millimeter relative to bregma according to the atlas of Paxinos and Watson (2007) were: AP, −2.2; L, −4.7; V, −8.3. One screw, placed above the left parietal cortex, served as the indifferent reference electrode. Additional skull screws and dental acrylic cement anchored the entire headset. After surgery, the animals were allowed to recover for periods of 12—55 days (see below). Following these periods, 183 rats were electrically stimulated over 25 min via the BLA electrode for induction of SSSE as previously described (Brandt et al., 2003). The stimulus consisted of 100 ms trains of 1 ms alternating positive and negative square wave pulses. The train frequency was 2 Hz and the intra-train pulse frequency was 50 Hz. The intensity of the stimulus was 700 A. In all rats, the EEG was recorded via the BLA electrode during SSSE. Four hours after the induction of the SSSE, the rats were injected with diazepam (10 mg/kg ip) to terminate the seizure activity. Sustained BLA stimulation induced three different types of SSSE, as previously reported (Brandt et al., 2003). Type 1, partial (nonconvulsive) SSSE, which was characterized by stage 1 and stage 2 seizures (Racine, 1972); type 2, partial SSSE interrupted by occasional periods of stage 3 and generalized convulsive (stage 4 or 5) seizures; type 3, generalized convulsive SSSE (for further details see Brandt et al., 2003). The typical paroxysmal EEG alterations occurring during these types of SSSE have been described in detail by us previously (see Fig. 1 in Brandt et al., 2003). Previous experiments by our group have shown that both type 2 and type 3 SSSE induce SRS within 2—3 months in >90% of female Sprague-Dawley rats and about the same extent of neuronal damage in the hippocampus, while after type 1 SSSE only about 30% of the rats develop SRS and neuronal damage is much less severe (Brandt et al., 2003). In addition to recording the different types of SSSE, the severity of SSSE was scored as follows: 0, no SE; 1, type 1; 3, type 2; 5, type 3, respectively. Based on recent studies in the kindling model (Löscher et al., 1993, 1995, 1999), the different intervals between electrode implantation and SE induction were arbitrarily grouped as follows: 12—21 days (group 1; n = 95; mean 16 ± 0.3 days), 22—34 days (group 2; n = 69; mean 28 ± 0.4 days), and 37—55 days (group 3; n = 19; mean 48 ± 1.6 days), respectively. The significance of differences in frequency of SE types between groups was calculated by Fisher’s exact test, whereas one-way ANOVA with posthoc Tukey—Kramer multiple comparisons test was used for statistical evaluation of score data. A P < 0.05 was considered significant. The authors were blinded to ‘‘time since stimulation’’ when they assessed the severity of the SSSE.
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Results As shown in Fig. 1A, with the previously used postsurgical interval of 2 weeks on average (Brandt et al., 2003), 9.4% of the rats of group 1 did not develop SSSE, 14.7% developed a type 1 SSSE, 27.4% a type 2 and 48.4% a type 3 SSSE, respectively. When rats with intervals of 4 weeks on average were examined, 5.8% of the 69 rats in group 2 did not develop SSSE, and 10.1, 40.6, and 43.5% a type 1, 2 or 3 SSSE, respectively, which was not significantly different from group 1 (Fig. 1A). At an interval of 7 weeks on average, none of the 19 rats in group 3 did not respond with SSSE to stimulation, only 5.3% exhibited a type 1 or 2 SSSE, but 89.5% responded with a type 3 SSSE. The low number of type 2 and high number of type 3 SSSE in group 3 were significantly different from the two other groups with shorter postsurgical intervals (Fig. 1A) A similar result was obtained when average SSSE severity scores were compared between the three groups (Fig. 1B). The group with the longest postsurgical interval (group 3) was also the group with the highest severity of SSSE.
Discussion In our previous prospective study in amygdala-kindled rats, kindling by once daily BLA stimulation was started after postsurgical recovery periods of either 1, 2, 4, or 8 weeks (Löscher et al., 1995). An enhanced kindling rate was seen when kindling stimulations were started after 4 and 8 weeks following electrode implantation. Furthermore, in fully kindled rats, the afterdischarge threshold (ADT) was significantly lower in the group with 8 weeks post-surgical delay. Based on these data, we proposed that prolonged implantation of an electrode into a sensitive region of the limbic system predisposes the brain to kindling (Löscher et al., 1995). In a subsequent study, Niespodziany et al. (1999) reported that prolonged implantation of a BLA electrode in rats resulted in a significant increase in the occurrence of repetitive population spikes in the CA3 area of hippocampal slices that was similar to the increase seen in BLA-kindled rats, suggesting that neuronal circuits in the hippocampal CA3 region represent a preferential locus of the epileptogenic consequences of prolonged electrode implantation in the amygdala. In the present study, electrodes that were in the BLA for more than 5 weeks (group 3) significantly increased the sensitivity of rats to SE induction. In contrast to our previous suggestion (Löscher et al., 1995), we do not believe that this finding is due to a prokindling effect of prolonged electrode implantation, because SE induction in previously BLA-kindled rats did not result in more severe types of SSSE than SE induction in control (non-kindled) rats (Brandt et al., 2003). Rather we believe that the present finding is due to epileptogenic consequences of prolonged electrode implantation in the amygdala, as previously suggested by Niespodziany et al. (1999). As described in the Introduction, likely explanations of this finding include the deposition of iron from hemoglobin destruction in local microhemorrhages caused by the implantation and consequences of local destruction of the blood-brain barrier resulting in extravasation of albumin, which needs to be further investigated.
Figure 1 Effects of prolonged implantation of a BLA electrode on types and severity of SE in rats. SE was induced by electrical stimulation via the bipolar BLA stimulation/recording electrode at average intervals of 2 (group 1; n = 95), 4 (group 2; n = 69), or 7 (group 3; n = 19) weeks after electrode implantation. In ‘‘A’’, the different SE types in each group are illustrated. In the group with the longest presence of the depth electrode (group 3), significantly less rats exhibited type 2 SE compared to group 1 (P = 0.0409) and group 2 (P = 0.0046). Furthermore, significantly more rats of group 3 exhibited the most severe type of SE, type 3, compared to group 1 (P = 0.0009) and group 2 (P = 0.0005). Groups 1 and 2 did not differ significantly. In ‘‘B’’, SE severity was scored as described in Methods. Data are illustrated as means + SEM of 95 (group 1), 69 (group 2) and 19 (group 3) rats, respectively. Analysis of data by ANOVA indicated a highly significant difference between groups (P = 0.008). Posthoc analysis indicated that group 3 significantly differed from group 1 (P < 0.01) and group 2 (P < 0.05).
Intracranial electrodes increase seizure susceptibility Liu et al. (2012) recently reported that depth electroderelated injuries in patients may lead to the generation of aberrant blood vessels, the upregulation of active inflammatory cell types and extravasation of plasma proteins into an area that is >30 times the area of the physical insult. Furthermore, we have described previously that prolonged BLA electrode implantation induces a significant decrease in GABA turnover in several brain regions, including amygdala and hippocampus (Löscher et al., 1999), which may contribute to the pro-epileptogenic effects. We do not believe that the differences in animals’ age at time of SE induction affected SE severity, because major differences in susceptibility to SE occur in developing vs. adult (Sankar et al., 1999; Sperber et al., 1999) and adult vs. aging rats (Liang et al., 2007) but not in the age span (about 95—130 days) used for SE induction in the present study. However, we can, of course, not exclude that increasing age played a role in the present findings. Although our observation does not specifically substantiate any mechanistic interpretation, it does highlight possible pitfalls when using and interpreting results deriving from rodents with prolonged implantation of depth electrodes. Because the recent clinical findings reported by Liu et al. (2012) demonstrate that depth electrode implantation induces local brain alterations, including inflammation and albumin extravasation, which are known to exert proepileptogenic effects, the present experimental findings suggesting such a proepileptogenic effect may be of clinical relevance and therefore should be reexamined by a prospective study standardizing animal age and intervals between procedure, as well as accompanying histological evaluation.
Acknowledgements We thank Christian Rathert and Nicole Ernst for skillful technical assistance. The study was supported by grants (Lo 274-10, Lo 274-11) from the Deutsche Forschungsgemeinschaft (Bonn, Germany).
References Blackwood, D.H.R., Martin, M.J., McQueen, J.K., 1982. Enhanced rate of kindling after prolonged electrode implantation into the amygdala of rats. J. Neurosci. Methods 5, 343—348. Boast, C.A., Reid, S.A., Johnson, P., Zornetzer, S.F., 1976. A caution to brain scientists: unsuspected hemorrhagic vascular damage resulting from mere electrode implantation. Brain Res. 103, 527—534.
805 Brandt, C., Glien, M., Potschka, H., Volk, H., Löscher, W., 2003. Epileptogenesis and neuropathology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats. Epilepsy Res. 55, 83—103. Friedman, A., Kaufer, D., Heinemann, U., 2009. Blood—brain barrier breakdown-inducing astrocytic transformation: novel targets for the prevention of epilepsy. Epilepsy Res. 85, 142—149. Liang, L.P., Beaudoin, M.E., Fritz, M.J., Fulton, R., Patel, M., 2007. Kainate-induced seizures, oxidative stress and neuronal loss in aging rats. Neuroscience 147, 1114—1118. Liu, J.Y., Thom, M., Catarino, C.B., Martinian, L., Figarella-Branger, D., Bartolomei, F., Koepp, M., Sisodiya, S.M., 2012. Neuropathology of the blood—brain barrier and pharmaco-resistance in human epilepsy. Brain 135, 3115—3133. Löscher, W., Brandt, C., 2010. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research. Pharmacol. Rev. 62, 668—700. Löscher, W., Hönack, D., Gramer, M., 1999. Effect of depth electrode implantation with or without subsequent kindling on GABA turnover in various rat brain regions. Epilepsy Res. 37, 95—108. Löscher, W., Hörstermann, D., Hönack, D., Rundfeldt, C., Wahnschaffe, U., 1993. Transmitter amino acid levels in rat brain regions after amygdala-kindling or chronic electrode implantation without kindling: evidence for a pro-kindling effect of prolonged electrode implantation. Neurochem. Res. 18, 775—781. Löscher, W., Wahnschaffe, U., Hönack, D., Rundfeldt, C., 1995. Does prolonged implantation of depth electrodes predispose the brain to kindling? Brain Res. 697, 197—204. Niespodziany, I., Klitgaard, H., Margineanu, D.G., 1999. Chronic electrode implantation entails epileptiform field potentials in rat hippocampal slices, similarly to amygdala kindling. Epilepsy Res. 36, 69—74. Paxinos, G., Watson, C., 2007. The rat brain in stereotaxic coordinates, 6th ed. Elsevier, Amsterdam. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32, 281—294. Sankar, R., Shin, D., Mazarati, A.M., Liu, H., Wasterlain, C.G., 1999. Ontogeny of self-sustaining status epilepticus. Dev. Neurosci. 21, 345—351. Sperber, E.F., Veliskova, J., Germano, I.M., Friedman, L.K., Moshe, S.L., 1999. Age-dependent vulnerability to seizures. Adv. Neurol. 79, 161—169. Van Loo, P., Carrette, E., Meurs, A., Goossens, L., Van Roost, D., Vonck, K., Boon, P., 2011. Surgical successes and failures of invasive video-EEG monitoring in the presurgical evaluation of epilepsy. Panminerva Med. 53, 227—240. Willmore, L.J., Sypert, G.W., Munson, J.B., Hurd, R.W., 1978. Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science 200, 1501—1502.
journal homepage: www.elsevier.com/locate/epilepsyres
SHORT COMMUNICATION
Prolonged depth electrode implantation in the limbic system increases the severity of status epilepticus in rats Jens P. Bankstahl a,b,1, Claudia Brandt a,b, Wolfgang Löscher a,b,∗ a
Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine, Hannover, Germany b Center for Systems Neuroscience, Hannover, Germany Received 28 August 2013; received in revised form 23 January 2014; accepted 29 January 2014 Available online 8 February 2014
KEYWORDS Epilepsy; Kindling; Seizures; Amygdala; Blood—brain barrier; Hemorrhage
Summary Chronically implanted intracranial depth electrodes are widely used for studying electroencephalographic activities in deep cerebral locations and for electrical stimulation of such locations. We have previously reported that prolonged implantation of an electrode in the basolateral amygdala (BLA) of rats facilitates subsequent kindling from this site, indicating a pro-kindling or pro-epileptogenic effect. To further characterize this phenomenon, we analyzed data from experiments in which we induced a self-sustaining status epilepticus (SSSE) by BLA stimulation following different periods of post-surgical delay. In a total of 183 Sprague-Dawley rats, three groups with different periods of postsurgical delay to onset of electrical stimulation were compared: group 1 (16 days on average), group 2 (28 days) and group 3 (48 days). Three types of SSSE were observed after BLA stimulation: type 1 (nonconvulsive), type 2 (nonconvulsive occasionally interrupted by generalized convulsive activity), and type 3 (generalized convulsive). While groups 1 and 2 did not differ in the frequency of these SSSE types, the group with the longest interval between electrode implantation and stimulation (group 3) showed significantly more severe SSSE than the two other groups. The data indicate that intracranial electrode implantation may increase the sensitivity of the implanted area to seizure induction, extending previous findings in the kindling model. Potential mechanisms of these findings include the functional consequences of local microhemorrhages and blood—brain barrier destruction. © 2014 Elsevier B.V. All rights reserved.
Abbreviations: ADT, afterdischarge threshold; BLA, basolateral amygdala; SE, status epilepticus; SRS, spontaneous recurrent seizures; SSSE, self-sustaning status epilepticus. ∗ Corresponding author at: Department of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine, Bünteweg 17, D30559 Hannover, Germany. Tel.: +49 511 856 8721; fax: +49 511 953 8581. E-mail address: [email protected] (W. Löscher). 1 Present address: Preclinical Molecular Imaging, Department of Nuclear Medicine, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. 0920-1211/$ — see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2014.01.025
Intracranial electrodes increase seizure susceptibility
Introduction It is generally assumed that the use of intracranial recording with depth electrodes in the presurgical evaluation of patients with medically intractable epilepsy is a reasonably safe method (Van Loo et al., 2011). Similarly, in experimental studies with chronic implantation of depth electrodes into specific brain regions for recording of electroencephalographic activity or electrical stimulation of discrete regions, it is often implicitly assumed that the implantation of electrodes has little if any effect on brain function and related behaviors. However, we and others have previously shown in rats that chronic implantation of electrodes constructed from a variety of materials into different parts of the brain may induce lasting neurochemical, neurophysiological, morphological, and behavioral alterations, including a prokindling effect (Blackwood et al., 1982; Löscher et al., 1993, 1995, 1999; Niespodziany et al., 1999). Boast et al. (1976) demonstrated in rodents that electrode implantation per se may result in functionally significant hemorrhagic vascular damage and consequent release of blood into brain tissue, leading to iron deposits, which may induce chronic focal epileptic discharges (Willmore et al., 1978). In epilepsy patients, the incidence of intracranial hemorrhage associated with depth electrodes is up to 14% (Van Loo et al., 2011). Furthermore, electrode implantation locally destroys the blood—brain barrier, which may lead to extravasation of albumin and, hence, development of an epileptic focus (Friedman et al., 2009). From these data, we recently hypothesized that kindling, i.e., a model in which repeated administration of initially subconvulsive electrical stimuli via a depth electrode in the limbic system induces seizures that progressively increase in severity and duration, may represent a model in which the consequences of traumatic brain injury (by electrode implantation) are facilitated by electrical stimulation (Löscher and Brandt, 2010). Over recent years, models in which a status epilepticus (SE) induced by electrical or chemical stimuli results in the development of spontaneous recurrent seizures (SRS) have progressively replaced kindling as a model of epileptogenesis (Löscher and Brandt, 2010). We have used such a model, in which a self-sustaining SE (SSSE) is induced by electrical stimulation of the anterior basolateral amygdala (BLA; Brandt et al., 2003), extensively over recent years and had repeatedly the impression that a long interval (≥4 weeks) between electrode implantation and SE induction increases the incidence and severity of SSSE. We therefore performed a retrospective analysis of data to determine whether prolonged electrode implantation in the limbic system alters the sensitivity of rats to SE induction.
Methods As in previous studies of our group on the SE model used in this study (Brandt et al., 2003), adult female SpragueDawley rats (body weight 200—230 g) were obtained from Harlan-Winkelmann (Borchen, Germany) or Harlan (Horst, Netherlands) and adapted for at least one week before onset of the experiments. Maintenance of rats was as previously described (Brandt et al., 2003). All experiments were
803 done in compliance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). All efforts were made to minimize pain or discomfort of the animals used. At time of electrode implantation, all rats were about 80 days of age. For electrode implantation, rats were anesthetized with chloral hydrate (360 mg/kg i.p.). A bipolar electrode was stereotaxically implanted into the right BLA and served as stimulation- and recording-electrode. As in recent studies of our group, the electrode consisted of two twisted Teflon-coated, 0.2 mm diameter, stainless steel wires separated by 0.5 mm at the tip. The stereotaxic coordinates in millimeter relative to bregma according to the atlas of Paxinos and Watson (2007) were: AP, −2.2; L, −4.7; V, −8.3. One screw, placed above the left parietal cortex, served as the indifferent reference electrode. Additional skull screws and dental acrylic cement anchored the entire headset. After surgery, the animals were allowed to recover for periods of 12—55 days (see below). Following these periods, 183 rats were electrically stimulated over 25 min via the BLA electrode for induction of SSSE as previously described (Brandt et al., 2003). The stimulus consisted of 100 ms trains of 1 ms alternating positive and negative square wave pulses. The train frequency was 2 Hz and the intra-train pulse frequency was 50 Hz. The intensity of the stimulus was 700 A. In all rats, the EEG was recorded via the BLA electrode during SSSE. Four hours after the induction of the SSSE, the rats were injected with diazepam (10 mg/kg ip) to terminate the seizure activity. Sustained BLA stimulation induced three different types of SSSE, as previously reported (Brandt et al., 2003). Type 1, partial (nonconvulsive) SSSE, which was characterized by stage 1 and stage 2 seizures (Racine, 1972); type 2, partial SSSE interrupted by occasional periods of stage 3 and generalized convulsive (stage 4 or 5) seizures; type 3, generalized convulsive SSSE (for further details see Brandt et al., 2003). The typical paroxysmal EEG alterations occurring during these types of SSSE have been described in detail by us previously (see Fig. 1 in Brandt et al., 2003). Previous experiments by our group have shown that both type 2 and type 3 SSSE induce SRS within 2—3 months in >90% of female Sprague-Dawley rats and about the same extent of neuronal damage in the hippocampus, while after type 1 SSSE only about 30% of the rats develop SRS and neuronal damage is much less severe (Brandt et al., 2003). In addition to recording the different types of SSSE, the severity of SSSE was scored as follows: 0, no SE; 1, type 1; 3, type 2; 5, type 3, respectively. Based on recent studies in the kindling model (Löscher et al., 1993, 1995, 1999), the different intervals between electrode implantation and SE induction were arbitrarily grouped as follows: 12—21 days (group 1; n = 95; mean 16 ± 0.3 days), 22—34 days (group 2; n = 69; mean 28 ± 0.4 days), and 37—55 days (group 3; n = 19; mean 48 ± 1.6 days), respectively. The significance of differences in frequency of SE types between groups was calculated by Fisher’s exact test, whereas one-way ANOVA with posthoc Tukey—Kramer multiple comparisons test was used for statistical evaluation of score data. A P < 0.05 was considered significant. The authors were blinded to ‘‘time since stimulation’’ when they assessed the severity of the SSSE.
804
J.P. Bankstahl et al.
Results As shown in Fig. 1A, with the previously used postsurgical interval of 2 weeks on average (Brandt et al., 2003), 9.4% of the rats of group 1 did not develop SSSE, 14.7% developed a type 1 SSSE, 27.4% a type 2 and 48.4% a type 3 SSSE, respectively. When rats with intervals of 4 weeks on average were examined, 5.8% of the 69 rats in group 2 did not develop SSSE, and 10.1, 40.6, and 43.5% a type 1, 2 or 3 SSSE, respectively, which was not significantly different from group 1 (Fig. 1A). At an interval of 7 weeks on average, none of the 19 rats in group 3 did not respond with SSSE to stimulation, only 5.3% exhibited a type 1 or 2 SSSE, but 89.5% responded with a type 3 SSSE. The low number of type 2 and high number of type 3 SSSE in group 3 were significantly different from the two other groups with shorter postsurgical intervals (Fig. 1A) A similar result was obtained when average SSSE severity scores were compared between the three groups (Fig. 1B). The group with the longest postsurgical interval (group 3) was also the group with the highest severity of SSSE.
Discussion In our previous prospective study in amygdala-kindled rats, kindling by once daily BLA stimulation was started after postsurgical recovery periods of either 1, 2, 4, or 8 weeks (Löscher et al., 1995). An enhanced kindling rate was seen when kindling stimulations were started after 4 and 8 weeks following electrode implantation. Furthermore, in fully kindled rats, the afterdischarge threshold (ADT) was significantly lower in the group with 8 weeks post-surgical delay. Based on these data, we proposed that prolonged implantation of an electrode into a sensitive region of the limbic system predisposes the brain to kindling (Löscher et al., 1995). In a subsequent study, Niespodziany et al. (1999) reported that prolonged implantation of a BLA electrode in rats resulted in a significant increase in the occurrence of repetitive population spikes in the CA3 area of hippocampal slices that was similar to the increase seen in BLA-kindled rats, suggesting that neuronal circuits in the hippocampal CA3 region represent a preferential locus of the epileptogenic consequences of prolonged electrode implantation in the amygdala. In the present study, electrodes that were in the BLA for more than 5 weeks (group 3) significantly increased the sensitivity of rats to SE induction. In contrast to our previous suggestion (Löscher et al., 1995), we do not believe that this finding is due to a prokindling effect of prolonged electrode implantation, because SE induction in previously BLA-kindled rats did not result in more severe types of SSSE than SE induction in control (non-kindled) rats (Brandt et al., 2003). Rather we believe that the present finding is due to epileptogenic consequences of prolonged electrode implantation in the amygdala, as previously suggested by Niespodziany et al. (1999). As described in the Introduction, likely explanations of this finding include the deposition of iron from hemoglobin destruction in local microhemorrhages caused by the implantation and consequences of local destruction of the blood-brain barrier resulting in extravasation of albumin, which needs to be further investigated.
Figure 1 Effects of prolonged implantation of a BLA electrode on types and severity of SE in rats. SE was induced by electrical stimulation via the bipolar BLA stimulation/recording electrode at average intervals of 2 (group 1; n = 95), 4 (group 2; n = 69), or 7 (group 3; n = 19) weeks after electrode implantation. In ‘‘A’’, the different SE types in each group are illustrated. In the group with the longest presence of the depth electrode (group 3), significantly less rats exhibited type 2 SE compared to group 1 (P = 0.0409) and group 2 (P = 0.0046). Furthermore, significantly more rats of group 3 exhibited the most severe type of SE, type 3, compared to group 1 (P = 0.0009) and group 2 (P = 0.0005). Groups 1 and 2 did not differ significantly. In ‘‘B’’, SE severity was scored as described in Methods. Data are illustrated as means + SEM of 95 (group 1), 69 (group 2) and 19 (group 3) rats, respectively. Analysis of data by ANOVA indicated a highly significant difference between groups (P = 0.008). Posthoc analysis indicated that group 3 significantly differed from group 1 (P < 0.01) and group 2 (P < 0.05).
Intracranial electrodes increase seizure susceptibility Liu et al. (2012) recently reported that depth electroderelated injuries in patients may lead to the generation of aberrant blood vessels, the upregulation of active inflammatory cell types and extravasation of plasma proteins into an area that is >30 times the area of the physical insult. Furthermore, we have described previously that prolonged BLA electrode implantation induces a significant decrease in GABA turnover in several brain regions, including amygdala and hippocampus (Löscher et al., 1999), which may contribute to the pro-epileptogenic effects. We do not believe that the differences in animals’ age at time of SE induction affected SE severity, because major differences in susceptibility to SE occur in developing vs. adult (Sankar et al., 1999; Sperber et al., 1999) and adult vs. aging rats (Liang et al., 2007) but not in the age span (about 95—130 days) used for SE induction in the present study. However, we can, of course, not exclude that increasing age played a role in the present findings. Although our observation does not specifically substantiate any mechanistic interpretation, it does highlight possible pitfalls when using and interpreting results deriving from rodents with prolonged implantation of depth electrodes. Because the recent clinical findings reported by Liu et al. (2012) demonstrate that depth electrode implantation induces local brain alterations, including inflammation and albumin extravasation, which are known to exert proepileptogenic effects, the present experimental findings suggesting such a proepileptogenic effect may be of clinical relevance and therefore should be reexamined by a prospective study standardizing animal age and intervals between procedure, as well as accompanying histological evaluation.
Acknowledgements We thank Christian Rathert and Nicole Ernst for skillful technical assistance. The study was supported by grants (Lo 274-10, Lo 274-11) from the Deutsche Forschungsgemeinschaft (Bonn, Germany).
References Blackwood, D.H.R., Martin, M.J., McQueen, J.K., 1982. Enhanced rate of kindling after prolonged electrode implantation into the amygdala of rats. J. Neurosci. Methods 5, 343—348. Boast, C.A., Reid, S.A., Johnson, P., Zornetzer, S.F., 1976. A caution to brain scientists: unsuspected hemorrhagic vascular damage resulting from mere electrode implantation. Brain Res. 103, 527—534.
805 Brandt, C., Glien, M., Potschka, H., Volk, H., Löscher, W., 2003. Epileptogenesis and neuropathology after different types of status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala in rats. Epilepsy Res. 55, 83—103. Friedman, A., Kaufer, D., Heinemann, U., 2009. Blood—brain barrier breakdown-inducing astrocytic transformation: novel targets for the prevention of epilepsy. Epilepsy Res. 85, 142—149. Liang, L.P., Beaudoin, M.E., Fritz, M.J., Fulton, R., Patel, M., 2007. Kainate-induced seizures, oxidative stress and neuronal loss in aging rats. Neuroscience 147, 1114—1118. Liu, J.Y., Thom, M., Catarino, C.B., Martinian, L., Figarella-Branger, D., Bartolomei, F., Koepp, M., Sisodiya, S.M., 2012. Neuropathology of the blood—brain barrier and pharmaco-resistance in human epilepsy. Brain 135, 3115—3133. Löscher, W., Brandt, C., 2010. Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research. Pharmacol. Rev. 62, 668—700. Löscher, W., Hönack, D., Gramer, M., 1999. Effect of depth electrode implantation with or without subsequent kindling on GABA turnover in various rat brain regions. Epilepsy Res. 37, 95—108. Löscher, W., Hörstermann, D., Hönack, D., Rundfeldt, C., Wahnschaffe, U., 1993. Transmitter amino acid levels in rat brain regions after amygdala-kindling or chronic electrode implantation without kindling: evidence for a pro-kindling effect of prolonged electrode implantation. Neurochem. Res. 18, 775—781. Löscher, W., Wahnschaffe, U., Hönack, D., Rundfeldt, C., 1995. Does prolonged implantation of depth electrodes predispose the brain to kindling? Brain Res. 697, 197—204. Niespodziany, I., Klitgaard, H., Margineanu, D.G., 1999. Chronic electrode implantation entails epileptiform field potentials in rat hippocampal slices, similarly to amygdala kindling. Epilepsy Res. 36, 69—74. Paxinos, G., Watson, C., 2007. The rat brain in stereotaxic coordinates, 6th ed. Elsevier, Amsterdam. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation: II. Motor seizure. Electroenceph. Clin. Neurophysiol. 32, 281—294. Sankar, R., Shin, D., Mazarati, A.M., Liu, H., Wasterlain, C.G., 1999. Ontogeny of self-sustaining status epilepticus. Dev. Neurosci. 21, 345—351. Sperber, E.F., Veliskova, J., Germano, I.M., Friedman, L.K., Moshe, S.L., 1999. Age-dependent vulnerability to seizures. Adv. Neurol. 79, 161—169. Van Loo, P., Carrette, E., Meurs, A., Goossens, L., Van Roost, D., Vonck, K., Boon, P., 2011. Surgical successes and failures of invasive video-EEG monitoring in the presurgical evaluation of epilepsy. Panminerva Med. 53, 227—240. Willmore, L.J., Sypert, G.W., Munson, J.B., Hurd, R.W., 1978. Chronic focal epileptiform discharges induced by injection of iron into rat and cat cortex. Science 200, 1501—1502.
