The Bumetanide-Sensitive Na-K-2Cl Cotransporter NKCC1 As a Potential Target of a Novel Mechanism-Based Treatment Strategy for Neonatal Seizures

Neurosurg Focus 25 (3):E22, 2008

The bumetanide-sensitive Na-K-2Cl cotransporter NKCC1 as a potential target of a novel mechanism-based treatment strategy for neonatal seizures

KRISTOPHER T. KAHLE, M.D., PH.D.,1 AND KEVIN J. STALEY, M.D.2 1Department of Neurosurgery and 2Division of Pediatric Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Seizures that occur during the neonatal period do so with a greater frequency than at any other age, have profound consequences for cognitive and motor development, and are difficult to treat with the existing series of antiepileptic drugs. During development, ␥-aminobutyric acid (GABA)ergic neurotransmission undergoes a switch from excitatory to – – inhibitory due to a reversal of neuronal chloride (Cl ) gradients. The intracellular level of chloride ([Cl ]i) in immature neonatal neurons, compared with mature adult neurons, is about 20-40 mM higher due to robust activity of the chlo-

ride-importing Na-K-2Cl cotransporter NKCC1, such that the binding of GABA to ligand-gated GABAA receptor- associated Cl– channels triggers Cl– efflux and depolarizing excitation. In adults, NKCC1 expression decreases and the – expression of the genetically related chloride-extruding K-Cl cotransporter KCC2 increases, lowering [Cl ]i to a level – such that activation of GABAA receptors triggers Cl influx and inhibitory hyperpolarization. The excitatory action of GABA in neonates, while playing an important role in neuronal development and synaptogenesis, accounts for the decreased seizure threshold, increased seizure propensity, and poor efficacy of GABAergic anticonvulsants in this age group. Bumetanide, a furosemide-related diuretic already used to treat volume overload in neonates, is a specific inhibitor of NKCC1 at low doses, can switch the GABA equilibrium potential of immature neurons from depolarizing to hyperpolarizing, and has recently been shown to inhibit epileptic activity in vitro and in vivo in animal models of neonatal seizures. The fundamental role of NKCC1 in establishing excitatory GABAergic neurotransmission in the neonate makes it a tempting target of a novel mechanism-based anticonvulsant strategy that could utilize the well- known pharmacology of bumetanide to help treat neonatal seizures. (DOI: 10.3171/FOC/2008/25/9/E22)

KEY WORDS • cation-chloride cotransporter • chloride transport • ␥-aminobutyric acid • KCC2 • NKCC1 • neonatal seizures

EIZURES occur more often during the neonatal period cluded that there is little evidence to endorse the use of any than at any other age (2–3.5 seizures per 1000 live of the anticonvulsants currently used in the neonatal peri- S births). Neonatal seizures have profound long-term od.6 The distinct clinical phenotype and treatment response consequences, with adult survivors often developing epi- of neonatal seizures, compared with adult seizures, is prob- lepsy and significant cognitive and motor disabilities.22 ably due to the different mechanisms of seizure generation, Nearly any neurological insult (such as hypoxia-ischemia, propagation, and termination in these 2 age groups. It is metabolic derangement, hemorrhage, and infection) sus- likely that an improved understanding of the molecular tained during this period can trigger the synchronous firing pathophysiology underlying neonatal seizures would lead of hyperexcitable neurons that underlie epileptogenesis.65 to improved treatment by defining targets for novel mech- Whereas phenobarbital, benzodiazepines, and phenytoin anism-based therapeutics. – have been the first-line treatments for neonatal seizures for In the mammalian CNS, the [Cl ]i in neurons determines years, these drugs are often ineffective and can even poten- the strength and polarity of GABAergic neurotransmission. 8,10,50 – tiate seizure episodes. A recent Cochrane Review con- The [Cl ]i is largely determined by the SLC12A cation-chloride cotransporters, including the Na-K-2Cl cotransporter NKCC1 (mediating Cl– entry) and the K-Cl cotransporter – 52,54 – KCC2 (mediating Cl exit). The mature neurons of Abbreviations used in this paper: [Cl ]i = intracellular concentra- – tion of chloride; CNS = central nervous system; E = chloride equi- adults exhibit low [Cl ]i due to minimal NKCC1 expression Cl but robust KCC2 expression, such that GABA-induced librium potential; EEG = electroencephalogram; EGABA = GABA equilibrium potential; GABA = ␥-aminobutyric acid; Ki = inhibi- allosteric activation of the GABAA receptor (which function constant; TLE = temporal lobe epilepsy. tions as a ligand-gated Cl– channel) results in an influx of

Neurosurg. Focus / Volume 25 / September 2008 1

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC K. T. Kahle and K. J. Staley negatively charged Cl– ions that triggers membrane hy- units, the initial diagnosis and treatment of seizures are of- perpolarization and synaptic inhibition. In adults, barbitu- ten based on clinical assessment alone, and EEGs are ob- rates and benzodiazepines decrease seizure activity by tained after the administration of antiepileptic drugs.61,65 increasing the duration and/or frequency of the opening of Unfortunately, it is an all-too-common occurrence for elec- – GABAA receptor-associated Cl channels. In contrast to the trographic seizures to persist in encephalopathic neonates mature neurons of adults, the immature neurons of neo- despite anticonvulsant drugs at “therapeutic” levels. – nates exhibit a much higher [Cl ]i due to robust NKCC1 Conventional antiepileptic drugs have limited utility in expression and minimal KCC2 expression, such that acti- treating neonatal seizures.6,28,41 Barbiturates and benzodi- – vation of GABAA receptors elicits an efflux of Cl ions that azepines, which are GABAA receptor agonists that are effi- triggers membrane depolarization and synaptic excitation. cacious for treating adult seizures, are currently among the The excitatory action of GABA in neonates plays a role in first-line drugs for neonatal seizures; however, they are neuronal development,2,3 but also accounts for their in- often ineffective, and have been shown to actually potenti- creased seizure propensity and lowered seizure threshold.68 ate seizure activity in the immature brain.19,67 Phenytoin has – 6,50 The high [Cl ]i of immature neurons also has a tendency to been used with a similar amount of success. Barbiturates render barbiturates and benzodiazepines ineffective, and benzodiazepines have also been known to produce a because these drugs (as compared with their action on phenomenon termed “electroclinical dissociation” in neo- – mature neurons, which harbor low [Cl ]i) facilitate the pas- nates, whereby the overt clinical manifestations of seizures sive outflow of Cl– down its electrochemical gradient, de- (convulsions) are inhibited, but electroencephalography- polarizing and exciting neurons.19,67 documented cortical seizure activity is either unaffected or Recent data have shown that the NKCC1 inhibitor bu- exacerbated.16,62 This insidious effect of the GABA agonists metanide, a drug already approved by the US Food and has the potential for great harm, because it provides physi- Drug Administration as a diuretic, shows efficacy against cians with a false sense of security that seizures are under seizures in the immature rodent brain, and thus may be clin- control, while cortical seizure activity—and its associated ically useful in the treatment of human neonatal sei- detrimental effects—continues. zures.19,21 This review will discuss the recent work that There have been few prospective studies or randomized has provided insight into the role of NKCC1 in fostering controlled trials of the antiepileptic drugs that are currently excitatory GABAergic neurotransmission in the immature used to treat neonatal seizures.6,12,60 To date, the only ran- brain, how the excitatory effect of GABA in neonates domized trial of antiepileptic drugs for the treatment of makes them particularly vulnerable to the development of neonatal seizures compared the current first-line drugs seizures, and how the pharmacological inhibition of (phenobarbital and phenytoin).50 In this study, the majority NKCC1 with bumetanide might hold promise for the treat- of neonates had asphyxia, infarction, or hemorrhage as the ment of seizures in neonates. origin of their seizures. Complete control of electrographic seizures was achieved with either drug in only ~ 25% of neonates whose seizure frequency was increasing. Seizure Neonatal Seizures Are a Common control was achieved in another 15% of newborns when Problem With Inadequate Treatment both agents were used concurrently. Preliminary studies of antiepileptic drugs other than phenobarbital and phenytoin Neonatal seizures, or epileptic episodes suffered by in- have shown only modest efficacy in smaller cohorts of fants in the first 28 days of life, occur in 1–2% of patients neonates, although sufficiently powered randomized trials in neonatal intensive care units, and are the most common are needed to conclusively demonstrate whether any manifestation of an acute neurological disorder in newborn 7,10,32,46,64 75 of these drugs are truly effective. The lack of evi- infants. Most commonly caused by hypoxic ischemic en- dence-based treatment recommendations, coupled with the cephalopathy, hemorrhage, or cerebral infarction, the pres- paucity of data regarding the underlying pathophysiology ence of neonatal seizures often portends severe neurologi- of neonatal seizures, has made their current management cal dysfunction later in life, with high rates of adult far from optimal. epilepsy and long-term cognitive and motor deficits in survivors.13,75 In animal models, neonatal seizures have been shown to be injurious to the development of the brain, in- The Strength and Polarity of GABAergic ducing synaptic reorganization, altering synaptic plasticity, Neurotransmission Are Determined by the and priming cortical neurons to increased damage from Intracellular Concentration of Chloride, Which Is seizures sustained later in life.57 Thus, the prompt diagnosis and successful treatment of neonatal seizures is important Established by NKCC1 and KCC2 for improving long-term neurological outcome. Recent research has provided information on key age- Whereas seizure activity in adults is usually clinically specific differences in GABAergic neurotransmission that obvious and the EEG reflects coordinated seizure activity, account for the decreased seizure threshold and lack of effi- diagnosing seizures in neonates is difficult because seizures cacy of conventional GABA agonists in neonates. Under- are often behaviorally subtle and the EEG typically demon- standing the molecular mechanisms that underlie the dif- strates a multifocal process.65 Nonetheless, seizures in neo- ferential actions of GABA in immature versus mature nates are currently diagnosed using electroencephalogra- neurons has, in turn, helped define targets for novel anti- phy, with a discharge duration of 10 seconds required to convulsants in neonates. diagnose an electrographic seizure (compared with 3 sec- In the adult cortex, GABA is the main inhibitory neuro- onds in older age groups). However, because EEGs are not transmitter, in which its activity is essential for maintaining immediately available in many neonatal intensive care appropriate electrical activity by balancing inputs that are

2 Neurosurg. Focus / Volume 25 / September 2008

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC Bumetanide and treatment strategies for neonatal seizures

FIG. 1. A switch in the neuronal expression of NKCC1 and KCC2 underlies the excitatory-to-inhibitory developmental shift in GABAergic signaling, in which GABAergic neurotransmission undergoes a switch from excitatory to inhibito- – – ry due to a reversal of neuronal Cl gradients during development. The ([Cl ]i) of immature neonatal neurons, compared with mature adult neurons, is much higher due to robust activity of the chloride-importing Na-K-2Cl cotransporter – – NKCC1, such that the binding of GABA to ligand-gated GABAA receptor-associated Cl channels triggers Cl efflux and depolarizing excitation. In adults, NKCC1 expression decreases and the expression of the genetically related chloride- – extruding K-Cl cotransporter KCC2 increases, lowering [Cl ]i to a level such that activation of GABAA receptors triggers Cl– influx and inhibitory hyperpolarization. The excitatory action of GABA in neonates accounts for the decreased seizure threshold and poor efficacy of GABAergic anticonvulsants in this age group. Reprinted with permission from Delpire E: News Physiol Sci 15:309–312, 2000.

3 – triggered by excitatory neurotransmitters such as gluta- mature neurons. Compared to mature adult neurons, [Cl ]i mate. In contrast, in the neonatal cortex, GABA is an exci- is ~ 20–40 mM higher in immature neonatal neurons, a dif- tatory neurotransmitter, where it plays an important role in ference that is sufficient to shift the action of GABA from neuronal development and the activity-dependent wiring of inhibition to excitation because the electrochemical driving circuits by triggering the depolarizing inputs that underlie force for Cl– drives the flux of this anion out of the cell 3 – large-scale spontaneous electrical activity. In immature when GABA channels are opened. The [Cl ]i is determined neurons, GABA-induced membrane depolarization trig- by the Na-K-Cl cotransporter NKCC1, which mediates Cl– gers action potentials that directly activate voltage-depen- transport into the cell, and the K-Cl cotransporter KCC2, dent Ca channels, and indirectly activate N-methyl- which mediates Cl– transport out of the cell. The NKCC1 D-aspartate receptors. The subsequent GABA-induced el- and KCC2 cotransporters are related members of the evations of intracellular Ca promote neuronal maturation, SLC12A electroneutral cation-chloride cotransporter gene and are important for the genesis and maintenance of syn- family.17,25,52 aptic connections.3 The cation-chloride cotransporters are intrinsic mem- – – Because the GABAA receptor is a ligand-gated Cl chan- brane proteins that transport Cl ions, together with Na and/ – nel, the resting level of [Cl ]i, and the ECl, are key determi- or K ions, across the plasma membranes of cells through- nants of GABA’s effect. Upon binding to GABAA re- out the body. They are targets of some of the most com- ceptors, GABA triggers conformational changes in monly used drugs in medicine (the “loop” and thiazide di- receptor-associated Cl– channels that permit either the pas- uretics), and are mutated in several inherited human sive influx or efflux of Cl– ions. The directionality of Cl– diseases, including the autosomal-recessive, renal, salt- – – 30 transport depends on the cell’s [Cl ]i. If the [Cl ]i is below wasting Gitelman and Bartter syndromes. The stoichio- – the ECl, Cl enters the cell and triggers membrane hyperpo- metric coupling and directionality of the ions translocated – larization and inhibition; if the [Cl ]i is greater than the ECl, by the cation-chloride cotransporters results in a secondar- Cl– enters the cell and triggers membrane depolarization ily active, nonelectrogenic (electroneutral) transport pro- and excitation. cess that is energetically driven by transmembrane Na and Recent work has shown that the differential action (exci- K gradients established by Na+-K+-adenosine triphospha- tatory vs inhibitory) of GABA in the neonate and adult tase. The cation-chloride cotransporters are divided into 2 – CNS is due to a difference in the [Cl ]i of immature versus main branches, defined by the identity and stoichiometry of

Neurosurg. Focus / Volume 25 / September 2008 3

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC K. T. Kahle and K. J. Staley

59 –/– their transported ions, their sensitivity to pharmacological EGABA, cortical neurons harvested from KCC2 mice fail – 79 inhibitors, and genetic phylogeny. to show a developmental decrease in [Cl ]i, and transgenic Given the large electrochemically favorable inward gra- overexpression of KCC2 in immature neurons abolishes dient for Na, the Na-coupled branch comprises the Na-(K)- the increases in intracellular Ca that are usually seen in Cl cotransporters NCC (the target of thiazide diuretics), early development as a result of GABA-induced membrane NKCC1, and NKCC2 (the target of Lasix, a furosemide depolarization.40 – – diuretic), which load Cl ions into the cell to raise [Cl ]i By acting as a self-limiting trophic factor, GABA might – above ECl. Conversely, by coupling the transport of Cl to itself trigger the developmental switch from NKCC1 to an outwardly-directed gradient for K, the 4 different K-Cl KCC2 by activating specific intracellular cascades that cotransporters (KCC1, KCC2, KCC3, and KCC4) pri- upregulate KCC2 gene expression.26 However, the devel- – – marily transport Cl ions out of the cell, lowering [Cl ]i opmental increase in KCC2 expression and the negative below ECl. The relative activities of the Na-(K)-Cl and K- shift in EGABA can take place in the presence of GABAA – Cl cotransporters determine [Cl ]i in numerous cell types, receptor antagonists, suggesting that GABA signaling may and their activities are reciprocally regulated by the chlo- be sufficient but not necessary for this transition.74 Recent ride-sensitive serine-threonine WNK and SPAK/OSR1 data suggest that spontaneous cholinergic activity, by trig- kinases.18,35,36 In neurons, NKCC1 and KCC2 are the cation- gering Ca-dependent signaling cascades that upregulate – 52 chloride cotransporters responsible for determining [Cl ]i. KCC2 expression downstream of the acetylcholine nico- Both bumetanide and furosemide, well-known loop di- tinic receptor, might also play a role in facilitating the exci- uretics, are capable of inhibiting the cation-chloride co- tatory-to-inhibitory GABAergic transition.42 transporters in vitro and in vivo.29,34 Bumetanide has an approximately 500-fold greater affinity for NKCC1 (K of ~ i Excitatory GABAergic Signaling in Immature 0.1 ␮M) than for KCC2 (K of ~ 25–50 ␮M). Furosemide i Neurons Predisposes Neonates to Seizures inhibits NKCC1 and KCC2 with equal potency (Ki of ~25- 50 ␮M). Therefore, at low doses (2–10 ␮M) bumetanide is Increasing synaptic excitation and/or decreasing synap- a relatively specific inhibitor of NKCC1.52 The accumula- tic inhibition can cause neurons to become hyperactive. tion of bumetanide in the CNS after systemic administra- Because neurons harbor a multiplicity of connections with tion has not been directly measured, but the drug’s high other neurons, the electrical firing of even a small popula- lipid:water partition coefficient and its documented anti- tion of hyperexcitable neurons, when synchronized, can convulsant effects in both animals and humans in vivo sug- progressively entrain larger neural networks until seizures gest that the drug is able to cross the blood–brain barri- ensue. In neonates, although GABA-mediated excitation er.19,21,66 plays a role in neuronal development, it also renders the developing brain particularly susceptible to seizures. In the A Switch in the Neuronal Expression of NKCC1 and adult cortex, excitatory glutaminergic signaling is balanced by inhibitory GABAergic signaling. However, in the KCC2 Underlies the Excitatory-to-Inhibitory neonate, the additional depolarization due to GABAA re- Developmental Shift in GABAergic Signaling ceptor activation likely adds to the excitation already initi- In embryonic and early postnatal life, there is robust neu- ated by glutamate neurotransmission to tip the balance of ronal expression of NKCC1, with minimal expression of excitation/inhibition toward excessive excitation and a KCC2.14,53,76 This predominance of inwardly directed Cl– propensity to seizure activity. Excitation mediated by – GABA has been shown to support epileptogenesis in the transport via NKCC1 increases [Cl ]i above ECl, such that GABA receptor activation triggers a passive outflow of developing hippocampus,20 and also to decrease the seizure A 38 Cl– ions that results in membrane depolarization and exci- threshold of neonates. The excitatory nature of GABA tation. Consistent with the important role of NKCC1 for signaling in immature neurons also explains why GABA agonists like barbiturates and benzodiazepines are often excitatory GABAergic neurotransmission, GABAA receptor-mediated depolarization is not present in NKCC1 ineffective in reducing neonatal seizures, and can even 50 mice,27 NKCC1 knockdown with RNA interference pro- exacerbate seizure activity. Clearly, new antiepileptic motes precocious inhibitory GABAergic signaling in treatment strategies are needed for neonatal seizures. immature neurons,9 and excitatory GABAergic signaling can be switched to inhibitory signaling in immature neu- Effect of Bumetanide, an Inhibitor of NKCC1, 58,69 rons by transgenic overexpression of KCC2. in Animal Models of Neonatal Seizures At birth, KCC2 is expressed at very low levels,59 and the – developmental negative shift in the GABA reversal poten- Because the elevated [Cl ]i of immature neurons is due to tial is paralleled by a robust increase in KCC2 expression robust activity of NKCC1, this cotransporter is currently near the end of the second postnatal week in rats.76 In hu- being explored as a target for novel anticonvulsant strate- mans, KCC2 expression begins at the 40th week after con- gies for neonatal seizures. In theory, inhibition of NKCC1, 21 – ception. This increase in KCC2 expression, accompanied by reducing [Cl ]i, could reduce the GABA-mediated exci- by the concurrent downregulation in NKCC1 expression, tation of immature neonatal neurons, or even possibly con- results in a dominance of Cl– export over Cl– import, de- vert the GABA response to an inhibitory one. – 21 creasing [Cl ]i below ECl. Consistent with the important role Dzhala et al. were the first to test this hypothesis in a of KCC2 for establishing inhibitory GABAergic neuro- recent seminal paper. Previous groups had established that transmission, knockdown of KCC2 in mature hippocampal NKCC1 expression in rats is highest in cortical neurons slices using RNA interference produces a positive shift in during the first postnatal week, begins to decrease at post-

4 Neurosurg. Focus / Volume 25 / September 2008

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC Bumetanide and treatment strategies for neonatal seizures natal Day 14, and then drops to the low levels that are vitro was employed. Such a model has the benefits of not found in adults.53,76 Conversely, the expression of KCC2 is altering the energy gradient for NKCC1 and preserving minimal at birth in rat cortical neurons, is low during the longitudinal intrahippocampal connections. Recurrent first postnatal week, and then attains an expression level at seizures were induced in the intact hippocampal prepara- postnatal Day 14 that is comparable with adults.44 In rats, tion by a continuous 5-hour exposure to low-magnesium the higher expression levels of NKCC1 earlier in develop- solution, and the anticonvulsant efficacy of phenobarbital, – 49,77 ment correlate with an increased [Cl ]i and with the pres- bumetanide, and the combination of these drugs was then ence of excitatory GABAergic signaling.3,20,39,48 Dzhala and studied. Whereas phenobarbital failed to abolish or depress colleagues21 demonstrated that a similar expression pattern recurrent seizures in 70% of immature hippocampi, pheno- is present in the human cortex, with high NKCC1 and low barbital in combination with bumetanide abolished seizures KCC2 expression during the neonatal time period and in 70% of immature hippocampi, and significantly reduced before the end of the 1st year of life, a time scale that is not the frequency, duration, and power of seizures in the re- significantly different from the scale in the rat when adjust- maining 30% of immature hippocampi. ed for the greater length of time required for the develop- Taken together, these in vitro and in vivo studies suggest ment of the human cortex relative to the rodent brain. These that bumetanide, alone or in combination with other drugs data supported the hypothesis that, similar to the rat, such as phenobarbital, might be useful in the treatment of GABA is excitatory in immature human cortical neurons, neonatal seizures in humans.19,21 and neonates may be susceptible to seizures due to the excitatory effects of GABA during development. Because – NKCC1 is known to establish the elevated levels of [Cl ]i Bridging the Gap From Animal Models that underlie excitatory GABAergic signaling in immature of Neonatal Seizures to Human Subjects neurons, these data predicted that bumetanide, an inhibitor of NKCC1, might be an effective treatment for neonatal At low concentrations (2–10 ␮M), bumetanide is a spe- seizures. cific inhibitor of NKCC1 and has well-established pharma- This hypothesis was tested by Staley67 in a series of ele- cokinetic and pharmacodynamic properties in adult hu- gant in vitro and in vivo physiological studies. These inves- mans with few side effects.68 Because the expression tigators found that NKCC1 blockade by bumetanide inhib- patterns of NKCC1 during development are similar in the ited cortical seizure activity in neonatal rats both in vitro human and rat cortex,21 bumetanide might be useful for the and in vivo, and this inhibition was observed at doses that treatment of seizures in human neonates. Bumetanide has have already been extensively tested in human neonates in been extensively used in both healthy and critically ill hu- diuresis studies.29 Specifically, they demonstrated that phar- man full-term and preterm infants to treat fluid volume macological inhibition of NKCC1 by bumetanide: 1) pro- overload due to cardiac and/or pulmonary disease, so ex- trapolation from these studies might help guide the design duced a negative shift in EGABA; 2) inhibited GABA-dependent synchronous excitatory activity in the immature of any potential pilot studies or clinical trials.43,72 However, hippocampus; 3) suppressed interictal and ictallike activity it will be important to investigate whether the pharmacoki- in immature hippocampal slices in vitro; and 4) attenuated netics of bumetanide are altered by any of the underlying kainate-induced seizure activity in vivo in neonatal rats. diseases that are responsible for triggering seizures in These anticonvulsant effects of bumetanide were shown to neonates, because many full-term newborns with refracto- be specific, as they were: 1) achieved at low doses that ry seizures have hypoxic-ischemic encephalopathy from selectively block NKCC1; 2) blocked by antagonists of the perinatal asphyxia, which is often accompanied by multi- GABA receptor, indicating that bumetanide is acting organ dysfunction (including hepatic and renal failure); A such organ dysfunction can dramatically affect drug metab- through a GABAA receptor-associated signaling pathway; 3) did not affect epileptiform activity in brain slices from olism.12,47,73 NKCC1–/– mice, indicating that inhibition of NKCC1 is the Perhaps the best new treatment strategy for neonatal sei- mechanism by which bumetanide exerts its GABA-depen- zures and one that could be easily tested, is a combination dent anticonvulsant effects; and 4) did not depress epilepti- regimen that would include bumetanide with a barbiturate form activity in mature neurons, in which the expression of like phenobarbital. In the neonatal rat brain, phenobarbital NKCC1 is Ͻ 10% of that in neonatal tissue.21 has been rendered a more effective anticonvulsant by coad- Given these promising findings, it seemed reasonable to ministering it with bumetanide, which reverses the Cl– gra- combine bumetanide, which blocks the excitatory effect of dient in immature neurons to a level such that GABAA – GABA in immature neurons by decreasing [Cl ]i, with phe- potentiation by phenobarbital results in synaptic inhibi- 19 nobarbital, a GABA agonist that opens GABAA receptor- tion. If such a trial were to take place, neonates with per- associated Cl– channels. Theoretically, such an increase in sistent seizures despite an initial loading dose of pheno- GABA-mediated conductance in neurons already targeted barbital (the current standard of care) could be offered by bumetanide would serve to increase shunting inhibition bumetanide along with the second dose of phenobarbital. and maximize the anticonvulsant power of the GABA sys- Continuous electroencephalography monitoring of patients tem. could then be used to determine whether bumetanide re- The efficacy of bumetanide, in combination with the duces seizures compared with controls (those neonates GABA-enhancing anticonvulsant phenobarbital, was re- treated with phenobarbital alone). Pilot studies now under- cently tested for the treatment of recurrent tonic-clonic way are examining the efficacy of bumetanide, adminis- epileptiform activity in the intact immature hippocampus in tered with phenobarbital, for the treatment of neonatal vitro.19 In this study, a low-magnesium model of neonatal seizures (FDA IND No. 101690; see http://www.cure seizures in the intact immature hippocampal formation in epilepsy.org/research/current.asp).

Neurosurg. Focus / Volume 25 / September 2008 5

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC K. T. Kahle and K. J. Staley

The combination of bumetanide and a barbiturate should NKCC1.63,71 These data suggest that a pathological increase obviate the need to use the high doses of barbiturate or ben- in NKCC1 activity due to an increase in cotransporter zodiazepine that have been associated with significant side phosphorylation contributes to the ischemia-induced accu- effects, such as apoptotic neurodegeneration in the devel- mulation of intracellular Cl– that triggers the neuronal hy- oping brain and late cognitive/behavioral impairment.4,5,23,56 perexcitability, excitotoxicity, and injury to neurons after Moreover, because of its longstanding safe use in newborns hypoxic-ischemic insults.11,54,78 and suggests that bumeta- as a diuretic, the low doses of bumetanide that are required nide might be useful for the treatment of seizures in this to inhibit NKCC1 are not anticipated to produce short- or clinical context. long-term side effects. However, caution must be exercised as work proceeds, and studies should be performed to References determine any potential side effects of inhibiting NKCC1 in the neonatal nervous system, because GABA-mediated 1. Beck J: Na-K-Cl cotransporter contributes to glutamate-mediated excitation is important for neuronal development.3 To date, excitotoxicity. J Neurosci 23:5061–5068, 2003 bumetanide-mediated inhibition of NKCC1 in the brain, 2. Ben-Ari Y: Developing networks play a similar melody. Trends for periods of time that would far exceed the duration that Neurosci 24:353–360, 2001 would be used for the treatment of neonatal seizures, has 3. Ben-Ari Y: Excitatory actions of GABA during development: the 9,27 nature of the nurture. Nat Rev Neurosci 3:728–739, 2002 been shown to have few developmental side effects. 4. Bittigau P, Sifringer M, Genz K, Reith E, Pospischil D, Govin- These modest but measurable side effects on cortical devel- darajalu S, et al: Antiepileptic drugs and apoptotic neurodegener- opment must be weighed against the well-known detri- ation in the developing brain. Proc Natl Acad Sci U S A 99: mental effects of persistent seizures in the immature brain 15089–15094, 2002 and the absence of knowledge regarding the developmen- 5. Bittigau P, Sifringer M, Ikonomidou C: Antiepileptic drugs and tal effects of NKCC1 inhibition in the setting of such apoptosis in the developing brain. Ann N Y Acad Sci 993: seizures.31 103–114, 123–124, 2003 6. Booth D, Evans DJ: Anticonvulsants for neonates with seizures. Cochrane Database Syst Rev: CD004218, 2004 Excitatory GABAergic Signaling 7. Boylan GB, Rennie JM, Chorley G, Pressler RM, Fox GF, Farrer K, et al: Second-line anticonvulsant treatment of neonatal sei- in Adult Epilepsy Syndromes zures: a video-EEG monitoring study. Neurology 62:486–488, Recent studies have also implicated excitatory GABA 2004 ergic signaling in the genesis of TLE and seizures that 8. Boylan GB, Rennie JM, Pressler RM, Wilson G, Morton M, 37,68 Binnie CD: Phenobarbitone, neonatal seizures, and video-EEG. occur after ischemic-hypoxemic insult in adults. A fea- Arch Dis Child Fetal Neonatal Ed 86:F165–F170, 2002 ture common to these seizure syndromes is that adult neu- 9. Cancedda L, Fiumelli H, Chen K, Poo MM: Excitatory GABA ac- rons adopt transmembrane Cl– gradients that phenotypical- tion is essential for morphological maturation of cortical neurons ly resemble those of neonatal neurons, rendering GABA in vivo. J Neurosci 27:5224–5235, 2007 activity excitatory instead of inhibitory.52 An increase in the 10. Castro Conde JR, Hernández Borges AA, Doménech Martínez E, relative abundance and/or activity of NKCC1 in relation to González Campo C, Perera Soler R: Midazolam in neonatal – seizures with no response to phenobarbital. Neurology 64: KCC2, which elevates [Cl ]i, accounts for this pathological shift. For example, neurons from individuals with adult 876–879, 2005 TLE accumulate Cl– in hippocampal neurons to an extent 11. Chen H, Sun D: The role of Na-K-Cl co-transporter in cerebral that renders GABA receptor activation excitatory,15 appar- ischemia. Neurol Res 27:280–286, 2005 A 12. Clancy RR: Summary proceedings from the neurology group on ently because of increased expression of NKCC1 and de- Pediatrics 117: 51 neonatal seizures. S23–S27, 2006 creased expression of KCC2. A human trial of bu- 13. Clancy RR, Legido A: Postnatal epilepsy after EEG-confirmed metanide is currently underway in adults with medically neonatal seizures. Epilepsia 32:69–76, 1991 intractable and surgically unresectable TLE associated with 14. Clayton GH, Owens GC, Wolff JS, Smith RL: Ontogeny of cat- mesial temporal sclerosis or different cortical malforma- ion-Cl– cotransporter expression in rat neocortex. Brain Res Dev tions.45 Brain Res 109:281–292, 1998 Bumetanide might also be useful for adult seizures that 15. Cohen I, Navarro V, Clemenceau S, Baulac M, Miles R: On the occur secondary to hypoxic-ischemic encephalopathy or origin of interictal activity in human temporal lobe epilepsy in vitro. Science 298:1418–1421, 2002 traumatic brain injury (due to brain edema), as NKCC1 is 16. Connell J, Oozeer R, de Vries L, Dubowitz LM, Dubowitz V: upregulated after both of these insults, and bumetanide has Clinical and EEG response to anticonvulsants in neonatal sei- been shown to decrease the excitability that accompanies zures. Arch Dis Child 64:459–464, 1989 – 52 the increases in neuronal [Cl ]i that follow these injuries. 17. Delpire E: Cation-chloride cotransporters in neuronal commu- For example, in rat models of cerebral hypoxic-ischemic nication. News Physiol Sci 15:309–312, 2002 – injury, a prolonged rise in [Cl ]i in hippocampal neurons 18. Delpire E, Gagnon KB: SPAK and OSR1: STE20 kinases in- renders GABAergic neurons hyperexcitable.24,33 This rise in volved in the regulation of ion homeostasis and volume control in – mammalian cells. Biochem J 409:321–331, 2008 [Cl ]i, which is completely prevented by bumetanide, is associated with an increase in the expression of the phos- 19. Dzhala VI, Brumback AC, Staley KJ: Bumetanide enhances phe- phorylated active form of NKCC1.55 Bumetanide has also nobarbital efficacy in a neonatal seizure model. Ann Neurol 63: been shown to prevent ischemia-induced hyperexcitability 222–235, 2008 1,70 20. Dzhala VI, Staley KJ: Excitatory actions of endogenously and cell swelling. The activation of glutamate receptors released GABA contribute to initiation of ictal epileptiform and high extracellular K, factors that contribute to the activity in the developing hippocampus. J Neurosci 23: pathogenesis of neuronal excitotoxicity, also have been 1840–1846, 2003 shown to induce the phosphorylation and activation of 21. Dzhala VI, Talos DM, Sdrulla DA, Brumback AC, Mathews GC,

6 Neurosurg. Focus / Volume 25 / September 2008

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC Bumetanide and treatment strategies for neonatal seizures

Benke TA, et al: NKCC1 transporter facilitates seizures in the pharmacokinetics of bumetanide in the newborn infant. Biol Neo- developing brain. Nat Med 11:1205–1213, 2005 nate 72:265–272, 1997 22. Evans D, Levene M: Neonatal seizures. Arch Dis Child Fetal 44. Lu J, Karadsheh M, Delpire E: Developmental regulation of the Neonatal Ed 78:F70–F75, 1998 neuronal-specific isoform of K-Cl cotransporter KCC2 in postna- 23. Farwell JR, Lee YJ, Hirtz DG, Sulzbacher SI, Ellenberg JH, Nel- tal rat brains. J Neurobiol 39:558–568, 1999 son KB: Phenobarbital for febrile seizures-effects on intelligence 45. Maa E, Bainbridge J, Spitz MC, Staley KJ: Oral bumetanide add- and on seizure recurrence. N Engl J Med 322:364–369, 1990 on therapy in refractory temporal lobe epilepsy. Epilepsia 48:S6, 24. Galeffi F, Sah R, Pond BB, George A, Schwartz-Bloom 2007 (Abstract) RD:Changes in intracellular chloride after oxygen-glucose depri- 46. Maytal J, Novak GP, King KC: Lorazepam in the treatment of vation of the adult hippocampal slice: effect of diazepam. J refractory neonatal seizures. J Child Neurol 6:319–323, 1991 Neurosci 24:4478–4488, 2004 47. McBride MC, Laroia N, Guillet R: Electrographic seizures in neo- 25. Gamba G: Molecular physiology and pathophysiology of elec- nates correlate with poor neurodevelopmental outcome. Neu- troneutral cation-chloride cotransporters. Physiol Rev 85:423– rology 55:506–513, 2000 493, 2005 48. Owens DF, Boyce LH, Davis MB, Kriegstein AR: Excitatory 26. Ganguly K, Schinder AF, Wong ST, Poo M: GABA itself pro- GABA responses in embryonic and neonatal cortical slices motes the developmental switch of neuronal GABAergic respons- demonstrated by gramicidin perforated-patch recordings and cal- es from excitation to inhibition. Cell 105:521–532, 2001 cium imaging. J Neurosci 16:6414–6423, 1996 27. Ge S, Goh EL, Sailor KA, Kitabatake Y, Ming GL, Song H: 49. Owens DF, Kriegstein AR: Is there more to GABA than synaptic GABA regulates synaptic integration of newly generated neurons inhibition? Nat Rev Neurosci 3:715–727, 2002 in the adult brain. Nature 439:589–593, 2006 50. Painter MJ, Scher MS, Stein AD, Armatti S, Wang Z, Gardiner JC, 28. Hakeem VF, Wallace SJ: EEG monitoring of therapy for neonatal et al: Phenobarbital compared with phenytoin for the treatment of seizures. Dev Med Child Neurol 32:858–864, 1990 neonatal seizures. N Engl J Med 341:485–489, 1999 29. Hannaert P, Alvarez-Guerra M, Pirot D, Nazaret C, Garay RP: Rat 51. Palma E, Amici M, Sobrero F, Spinelli G, Di Angelantonio S, NKCC2/NKCC1 cotransporter selectivity for loop diuretic drugs. Ragozzino D, et al: Anomalous levels of Cl– transporters in the Naunyn Schmiedebergs Arch Pharmacol 365:193–199, 2002 hippocampal subiculum from temporal lobe epilepsy patients 30. Hebert SC, Mount DB, Gamba G: Molecular physiology of make GABA excitatory. Proc Natl Acad Sci U S A 103:8465– cation-coupled Cl– cotransport: the SLC12 family. Pflugers Arch 8468, 2006 447:580–593, 2004 52. Payne JA, Rivera C, Voipio J, Kaila K: Cation-chloride co-trans- 31. Holmes GL, Ben-Ari Y. Seizures in the developing brain: perhaps porters in neuronal communication, development and trauma. not so benign after all. Neuron 21:1231–1234, 1998 Trends Neurosci 26:199–206, 2003 32. Hoppen T, Elger CE, Bartmann P: Carbamazepine in phenobarbi- 53. Plotkin MD, Snyder EY, Hebert SC, Delpire E: Expression of the tal-nonresponders: experience with ten preterm infants. Eur J Pe- Na-K-2Cl cotransporter is developmentally regulated in postnatal diatr 160:444–447, 2001 rat brains: a possible mechanism underlying GABA’s excitatory 33. Inglefield JR, Schwartz-Bloom RD: Activation of excitatory role in immature brain. J Neurobiol 33:781–795, 1997 amino acid receptors in the rat hippocampal slice increases intra- 54. Pond BB, Berglund K, Kuner T, Feng G, Augustine GJ, Schwartz- cellular Cl– and cell volume. J Neurochem 71:1396–1404, 1998 Bloom RD: The chloride transporter Na(+)-K(+)-Cl– cotrans- 34. Isenring P, Forbush B: Ion transport and ligand binding by the Na- porter isoform-1 contributes to intracellular chloride increases K-Cl cotransporter, structure-function studies. Comp Biochem after in vitro ischemia. J Neurosci 26:1396–1406, 2006 Physiol A Mol Integr Physiol 130:487–497, 2001 55. Pond BB, Galeffi F, Ahrens R, Schwartz-Bloom RD: Chloride 35. Kahle KT, Rinehart J, de Los Heros P, Louvi A, Meade P, transport inhibitors influence recovery from oxygen-glucose Vazquez N, et al: WNK3 modulates transport of Cl– in and out deprivation-induced cellular injury in adult hippocampus. Neu- of cells: implications for control of cell volume and neuronal ropharmacology 47:253–256, 2004 excitability. Proc Natl Acad Sci U S A 102:16783–16788, 56. Reinisch JM, Sanders SA, Mortensen EL, Rubin DB: In utero 2005 exposure to phenobarbital and intelligence deficits in adult men. 36. Kahle KT, Rinehart J, Ring A, Gimenez I, Gamba G, Hebert SC, JAMA 274:1518–1525, 1995 et al: WNK protein kinases modulate cellular Cl– flux by altering 57. Rennie JM, Boylan GB: Neonatal seizures and their treatment. the phosphorylation state of the Na-K-Cl and K-Cl cotransporters. Curr Opin Neurol 16:177–181, 2003 Physiology (Bethesda) 21:326–335, 2006 58. Reynolds A, Brustein E, Liao M, Mercado A, Babilonia E, Mount 37. Kahle KT, Staley K: Altered neuronal chloride homeostasis and DB, et al: Neurogenic role of the depolarizing chloride gradient excitatory GABAergic signaling in human temporal lobe epilep- revealed by global overexpression of KCC2 from the onset of sy. Epilepsy Curr 8:51–53, 2008 development. J Neurosci 28:1588–1597, 2008 38. Khalilov I, Holmes GL, Ben-Ari Y: In vitro formation of a sec- 59. Rivera C, Voipio J, Payne JA, Ruusuvuori E, Lahtinen H, Lamsa ondary epileptogenic mirror focus by interhippocampal propaga- K, et al: The K+/Cl– co-transporter KCC2 renders GABA hyper- tion of seizures. Nat Neurosci 6:1079–1085, 2003 polarizing during neuronal maturation. Nature 397:251–255, 39. Khazipov R, Khalilov I, Tyzio R, Morozova E, Ben-Ari Y, Hol- 1999 mes GL: Developmental changes in GABAergic actions and 60. Sankar R, Painter MJ: Neonatal seizures: after all these years we seizure susceptibility in the rat hippocampus. Eur J Neurosci 19: still love what doesn’t work. Neurology 64:776–777, 2005 590–600, 2004 61. Scher MS: Seizures in the newborn infant. Diagnosis, treatment, 40. Lee H, Chen CX, Liu YJ, Aizenman E, Kandler K: KCC2 expres- and outcome. Clin Perinatol 24:735–772, 1997 sion in immature rat cortical neurons is sufficient to switch the 62. Scher MS, Alvin J, Gaus L, Minnigh B, Painter MJ: Uncoupling polarity of GABA responses. Eur J Neurosci 21:2593–2599, of EEG-clinical neonatal seizures after antiepileptic drug use. 2005 Pediatr Neurol 28:277–280, 2003 41. Legido A, Clancy RR, Berman PH: Neurologic outcome after 63. Schomberg SL, Su G, Haworth RA, Sun D: Stimulation of Na-K- electroencephalographically proven neonatal seizures. Pediatrics 2Cl cotransporter in neurons by activation of non-NMDA 88:583–596, 1991 ionotropic receptor and group-I mGluRs. J Neurophysiol 85: 42. Liu Z, Neff RA, Berg DK: Sequential interplay of nicotinic and 2563–2575, 2001 GABAergic signaling guides neuronal development. Science 64. Sheth RD, Buckley DJ, Gutierrez AR, Gingold M, Bodensteiner 314:1610–1613, 2006 JB, Penney S: Midazolam in the treatment of refractory neonatal 43. Lopez-Samblas AM, Adams JA, Goldberg RN, Modi MW: The seizures. Clin Neuropharmacol 19:165–170, 1996

Neurosurg. Focus / Volume 25 / September 2008 7

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC K. T. Kahle and K. J. Staley

65. Silverstein FS, Jensen FE: Neonatal seizures. Ann Neurol 62: Hyperpolarizing inhibition develops without trophic support by 112–120, 2007 GABA in cultured rat midbrain neurons. J Physiol 550:719–730, 66. Sipilä ST, Schuchmann S, Voipio J, Yamada J, Kaila K: The 2003 cation-chloride cotransporter NKCC1 promotes sharp waves in 75. Volpe JJ: Neurology of the Newborn. Philadelphia: WB the neonatal rat hippocampus. J Physiol 573:765–773, 2006 Saunders, 2001, pp 178–214 67. Staley K: Enhancement of the excitatory actions of GABA by bar- 76. Wang C, Shimizu-Okabe C, Watanabe K, Okabe A, Matsuzaki H, biturates and benzodiazepines. Neurosci Lett 146:105–107, 1992 Ogawa T, et al: Developmental changes in KCC1, KCC2, and 68. Staley KJ: Wrong-way chloride transport: is it a treatable cause of NKCC1 mRNA expressions in the rat brain. Brain Res Dev some intractable seizures? Epilepsy Currents 6:124–127, 2006 Brain Res 139:59–66, 2002 69. Stein V, Hermans-Borgmeyer I, Jentsch TJ, Hübner CA: Ex- 77. Yamada J, Okabe A, Toyoda H, Kilb W, Luhmann HJ, Fukuda A: pression of the KCl cotransporter KCC2 parallels neuronal matu- Cl– uptake promoting depolarizing GABA actions in immature rat ration and the emergence of low intracellular chloride. J Comp neocortical neurones is mediated by NKCC1. J Physiol Neurol 468:57–64, 2004 557:829–841, 2004 70. Su G, Kintner DB, Flagella M, Shull GE, Sun D: Astrocytes from 78. Yan Y, Dempsey RJ, Flemmer A, Forbush B, Sun D: Inhibition of Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of Na(+)-K(+)-Cl(-) cotransporter during focal cerebral ischemia swelling and decrease in EAA release. Am J Physiol Cell Physiol decreases edema and neuronal damage. Brain Res 961:22–31, 282:C1147–C1160, 2002 2003 71. Su G, Kintner DB, Sun D: Contribution of Na(+)-K(+)-Cl(-) 79. Zhu L, Lovinger D, Delpire E: Cortical neurons lacking KCC2 cotransporter to high-[K(+)](o)-induced swelling and EAA expression show impaired regulation of intracellular chloride. J release in astrocytes. Am J Physiol Cell Physiol 282:C1136– Neurophysiol 93:1557–1568, 2005 C1146, 2002 72. Sullivan JE, Witte MK, Yamashita TS, Myers CM, Blumer JL: Pharmacokinetics of bumetanide in critically ill infants. Clin Pharmacol Ther 60:405–413, 1996 Manuscript submitted May 15, 2008. 73. Tekgul H, Gauvreau K, Soul J, Murphy L, Robertson R, Stewart Accepted June 25, 2008. J, et al: The current etiologic profile and neurodevelopmental out- Address correspondence to: Kristopher T. Kahle, M.D., Ph.D., come of seizures in term newborn infants. Pediatrics 117:1270– Department of Neurosurgery, Massachusetts General Hospital, Har- 1280, 2006 vard Medical School, 55 Fruit Street, Boston, Massachusetts 02114. 74. Titz S, Hans M, Kelsch W, Lewen A, Swandulla D, Misgeld U: email: [email protected].

8 Neurosurg. Focus / Volume 25 / September 2008

Unauthenticated | Downloaded 10/02/21 12:08 PM UTC