- Email: [email protected]
Response by the authors
Epilepsy Research 71 (2006) 239–240
Reply to Letter to the Editor Response by the authors Dear Sir, Frey’s (2006) comment on our recent paper (Margineanu and Klitgaard, 2006) essentially revolves around an alleged excessiveness of drug concentrations we applied in the perfusion fluid of the rat hippocampal slices, given that those concentrations in the plasma would be toxic in vivo. While not disputing in any way the putative toxicity of loop diuretics when their concentrations in the plasma would be in the ∼mM range, we stress having carefully avoided any mix-up between the effects we observed on hippocampal slices (in vitro) and putative antiepileptic activity in vivo. Moreover, we deem interesting Frey’s suggestion that the antiseizure effect of loop diuretics in audiogenic seizure-prone rats (Reid et al., 2000) might relate to an ototoxic effect and we take note of his strong statement that an antiepileptic effect of loop diuretics might only be induced in in vitro experiments, though this is at odds with repeated medical accounts (e.g. Hesdorffer et al., 2001; Haglund and Hochman, 2005). However, since from the title up to the concluding paragraph we repeatedly reminded the in vitro type of model on which the reported results were observed, and we did not make any shortcut to in vivo activity, we have to observe that Frey’s (2006) comments simply do not relate to the aim of our study, which is to further characterize the long known antiepileptic activity in vitro of cation-chloride co-transport-blocking diuretics. As for the drug concentrations we used, including the mM concentrations of furosemide, they are as used in all the studies reporting effects on neurons in vitro, including – but not restricted to – those we quoted in Methods of our paper. On this line, the nearly absence of effect of 0920-1211/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2006.06.006
bumetanide in our model, cannot be attributed to having used too low (!) concentrations of this drug, since Fig. 2(C) shows a tendency of the upper concentration of bumetanide to increase spontaneous bursting. Moreover, the very absence of effect in vivo of high doses of bumetanide, quoted by Frey (Østergaard et al., 1972), appears in agreement with the lack of effect we reported. Beyond the disappointment that Frey (2006) simply ignores the mechanistic message of our study, we notice the rather superficial and biased reading of our paper, on which his comment is based. Thus, the first paragraph of the comment reads that a “postulated” (!) antiepileptic effect would have been observed only with the highest concentration tested and after 95 min of superfusion. However, the graph in Fig. 2(A) of our paper clearly shows a concentration-dependent inhibition of the spontaneous bursting by three increasing concentrations of ethacrynic acid, which was quite evident within 40 min of drug addition to the perfusion fluid. The commentator hastily took the indications on the abscissas in Fig. 2 as representing the duration of drug superfusion, overlooking the fact that the drugs were added to the perfusion of the slices only when the epileptiform markers were fully expressed, 40 min after shifting from the normal artificial cerebrospinal fluid to the epileptogenic high K+ –low Ca2+ perfusion fluid. However, this is not only stated in Methods, but also clearly marked on both Figs. 2 and 3. We do not plainly refute the possibility that other effects of the diuretics than cation-chloride co-transport blockade might also contribute to their antiepileptic activity in vitro, so much since we even quoted in Discussion a paper (Bonnet et al., 1996) showing other effects of the ethacrynic acid—the compound with best expressed activity in our study. However, we do con-
240
Reply to Letter to the Editor / Epilepsy Research 71 (2006) 239–240
sider the report by Dzhala et al. (2005) that bumetanide strongly depressed epileptiform activity in hippocampal slices from rats up to P12, but was ineffective in P21–23 slices (as it was in our slices from 5-weeks-old rats) as highly relevant and supportive of the interpretation we proposed. Summing up, we deem useful considering Frey’s (2006) comment as a reminder that in vitro results should not be indiscriminately equated with in vivo activity, a mistake not present in our paper.
References Bonnet, U., Gastpar, M., Bingmann, D., 1996. Ethacrynic acid: effects on postsynaptic GABA responses and electric activity of CA3 neurones. Neuroreport 7, 2983–2987. Dzhala, V., Talos, D.M., Sdrulla, D.A., Brumback, A.C., Mathews, G.C., Benke, T.A., Delpire, E., Jensen, F.E., Staley, K.J., 2005. NKCC1 transporter facilitates seizures in the developing brain. Nat. Med. 11, 1205–1213. Frey, H.-H., 2006. Antiepileptic effect of loop diuretics? (Letter to the Editors). Epilepsy Res., doi:10.1016/j.eplepsyres.2006.06.005, in press. Haglund, M.M., Hochman, D.W., 2005. Furosemide and mannitol suppression of epileptic activity in the human brain. J. Neurophysiol. 94, 907–918.
Hesdorffer, D.C., Stables, J.P., Hauser, W.A., Annegers, J.F., Cascino, G., 2001. Are certain diuretics also anticonvulsants? Ann. Neurol. 50, 458–462. Margineanu, D.G., Klitgaard, H., 2006. Differential effects of cation-chloride co-transport-blocking diuretics in a rat hippocampal slice model of epilepsy. Epilepsy Res. 69, 93– 99. Østergaard, E.H., Magnussen, M.P., Kærgaard Nielsen, C., Eilertsen, E., Frey, H.-H., 1972. Pharmacological properties of 3-nbutylamino-4-phenoxy-5-sulfamylbenzoic acid (bumetanide), a new potent diuretic. Arzneimittelforschung 22, 66–72. Reid, K.H., Guo, S.Z., Iyer, V.G., 2000. Agents which block potassium-chloride cotransport prevent sound-triggered seizures in post-ischemic audiogenic seizure-prone rats. Brain Res. 864, 134–137.
Doru Georg Margineanu ∗ Henrik Klitgaard UCB S.A., CNS Research, B-1420 Braine-l’Alleud, Belgium ∗ Corresponding
author. Tel.: +32 2 386 26 37; fax: +32 2 386 33 97. E-mail address: [email protected] 15 June 2006 Available online 28 July 2006
Reply to Letter to the Editor Response by the authors Dear Sir, Frey’s (2006) comment on our recent paper (Margineanu and Klitgaard, 2006) essentially revolves around an alleged excessiveness of drug concentrations we applied in the perfusion fluid of the rat hippocampal slices, given that those concentrations in the plasma would be toxic in vivo. While not disputing in any way the putative toxicity of loop diuretics when their concentrations in the plasma would be in the ∼mM range, we stress having carefully avoided any mix-up between the effects we observed on hippocampal slices (in vitro) and putative antiepileptic activity in vivo. Moreover, we deem interesting Frey’s suggestion that the antiseizure effect of loop diuretics in audiogenic seizure-prone rats (Reid et al., 2000) might relate to an ototoxic effect and we take note of his strong statement that an antiepileptic effect of loop diuretics might only be induced in in vitro experiments, though this is at odds with repeated medical accounts (e.g. Hesdorffer et al., 2001; Haglund and Hochman, 2005). However, since from the title up to the concluding paragraph we repeatedly reminded the in vitro type of model on which the reported results were observed, and we did not make any shortcut to in vivo activity, we have to observe that Frey’s (2006) comments simply do not relate to the aim of our study, which is to further characterize the long known antiepileptic activity in vitro of cation-chloride co-transport-blocking diuretics. As for the drug concentrations we used, including the mM concentrations of furosemide, they are as used in all the studies reporting effects on neurons in vitro, including – but not restricted to – those we quoted in Methods of our paper. On this line, the nearly absence of effect of 0920-1211/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2006.06.006
bumetanide in our model, cannot be attributed to having used too low (!) concentrations of this drug, since Fig. 2(C) shows a tendency of the upper concentration of bumetanide to increase spontaneous bursting. Moreover, the very absence of effect in vivo of high doses of bumetanide, quoted by Frey (Østergaard et al., 1972), appears in agreement with the lack of effect we reported. Beyond the disappointment that Frey (2006) simply ignores the mechanistic message of our study, we notice the rather superficial and biased reading of our paper, on which his comment is based. Thus, the first paragraph of the comment reads that a “postulated” (!) antiepileptic effect would have been observed only with the highest concentration tested and after 95 min of superfusion. However, the graph in Fig. 2(A) of our paper clearly shows a concentration-dependent inhibition of the spontaneous bursting by three increasing concentrations of ethacrynic acid, which was quite evident within 40 min of drug addition to the perfusion fluid. The commentator hastily took the indications on the abscissas in Fig. 2 as representing the duration of drug superfusion, overlooking the fact that the drugs were added to the perfusion of the slices only when the epileptiform markers were fully expressed, 40 min after shifting from the normal artificial cerebrospinal fluid to the epileptogenic high K+ –low Ca2+ perfusion fluid. However, this is not only stated in Methods, but also clearly marked on both Figs. 2 and 3. We do not plainly refute the possibility that other effects of the diuretics than cation-chloride co-transport blockade might also contribute to their antiepileptic activity in vitro, so much since we even quoted in Discussion a paper (Bonnet et al., 1996) showing other effects of the ethacrynic acid—the compound with best expressed activity in our study. However, we do con-
240
Reply to Letter to the Editor / Epilepsy Research 71 (2006) 239–240
sider the report by Dzhala et al. (2005) that bumetanide strongly depressed epileptiform activity in hippocampal slices from rats up to P12, but was ineffective in P21–23 slices (as it was in our slices from 5-weeks-old rats) as highly relevant and supportive of the interpretation we proposed. Summing up, we deem useful considering Frey’s (2006) comment as a reminder that in vitro results should not be indiscriminately equated with in vivo activity, a mistake not present in our paper.
References Bonnet, U., Gastpar, M., Bingmann, D., 1996. Ethacrynic acid: effects on postsynaptic GABA responses and electric activity of CA3 neurones. Neuroreport 7, 2983–2987. Dzhala, V., Talos, D.M., Sdrulla, D.A., Brumback, A.C., Mathews, G.C., Benke, T.A., Delpire, E., Jensen, F.E., Staley, K.J., 2005. NKCC1 transporter facilitates seizures in the developing brain. Nat. Med. 11, 1205–1213. Frey, H.-H., 2006. Antiepileptic effect of loop diuretics? (Letter to the Editors). Epilepsy Res., doi:10.1016/j.eplepsyres.2006.06.005, in press. Haglund, M.M., Hochman, D.W., 2005. Furosemide and mannitol suppression of epileptic activity in the human brain. J. Neurophysiol. 94, 907–918.
Hesdorffer, D.C., Stables, J.P., Hauser, W.A., Annegers, J.F., Cascino, G., 2001. Are certain diuretics also anticonvulsants? Ann. Neurol. 50, 458–462. Margineanu, D.G., Klitgaard, H., 2006. Differential effects of cation-chloride co-transport-blocking diuretics in a rat hippocampal slice model of epilepsy. Epilepsy Res. 69, 93– 99. Østergaard, E.H., Magnussen, M.P., Kærgaard Nielsen, C., Eilertsen, E., Frey, H.-H., 1972. Pharmacological properties of 3-nbutylamino-4-phenoxy-5-sulfamylbenzoic acid (bumetanide), a new potent diuretic. Arzneimittelforschung 22, 66–72. Reid, K.H., Guo, S.Z., Iyer, V.G., 2000. Agents which block potassium-chloride cotransport prevent sound-triggered seizures in post-ischemic audiogenic seizure-prone rats. Brain Res. 864, 134–137.
Doru Georg Margineanu ∗ Henrik Klitgaard UCB S.A., CNS Research, B-1420 Braine-l’Alleud, Belgium ∗ Corresponding
author. Tel.: +32 2 386 26 37; fax: +32 2 386 33 97. E-mail address: [email protected] 15 June 2006 Available online 28 July 2006