Water movement in brain tissue
Human plasma osmolality is homeostatically maintained at 287 mosmol/kg H20 (mOsm), varying by ±1%. However, acute hyperosmolality (and the resulting “shrinkage” of cell volume) promotes the reactive intracellular accumulation of Na+, K+, and Cl− via regulatory volume increase (RVI); a more chronic exposure to hyperosmolar conditions causes brain cells to generate “idiogenic osmoles” (primarily free amino acids) to combat cell shrinkage (Hoffmann et al., 2009) (Fig. 1). Acute hypoosmolality promotes the transport of Na+, K+, and Cl− out of cells via regulatory volume decrease (RVD), and the loss of intracellular “idiogenic osmoles” in order to combat cell swelling. However, hyperacute osmotic changes that occur within minutes are not buffered by these adaptive mechanisms but instead allow rapid water shifts across membranes between cells (neurons and glia) and the extracellular space (ECS), resulting in rapid cell swelling in hypotonic ECS and cell shrinkage in hypertonic ECS (Andrew, 1991). Glial cells typically show faster rates of volume change because they contain aquaporins, whereas neurons do not (Amiry-Moghaddam & Ottersen, 2003; Tait et al., 2008).
Cerebral hydration and seizures
In studies of water intoxication in 1926, Rowntree administered pituitary extract (adrenocorticotropic hormone, ADH) with increasing amounts of free water and induced generalized seizures in animals, which were prevented by injection of hypertonic NaCl in follow-up experiments.
Carter described the experience of his group using hypertonic urea to treat status epilepticus in institutionalized patients (1962). Based on a prior series of more than 1,000 cases of status epilepticus, their use of barbiturates controlled status within 10 min, but required an average of four injections or 24 h of treatment to maintain seizure control. Their barbiturate therapy success rate was 90% but with 10% mortality. In the hypertonic urea series, they infused 57 patients with intravenous urea (half of the patients had previously received a standard barbiturate dose but continued seizing). Status epilepticus was stopped in all 57 patients, 51 patients having stoppage between 3 and 6 min, the longest amount of time being 10 min. There was no mortality in this series (1962).
Urea, a water-soluble and poorly membrane-permeable compound, increased the electroshock threshold of rats in studies by Reed and Woodbury, who felt its mechanism “is probably related more closely to the shifts of water and electrolytes between the intracellular and extracellular compartments of the brain than to the gross changes in total brain water and electrolytes” (1964). They concluded from these findings that hyposmolar conditions and resultant cell swelling and ECS shrinkage appear to promote seizures, whereas hyperosmolar conditions and resultant cell shrinkage and ECS expansion appear to prevent seizures (1964).
Because of their interest in the brain edema that accompanies kainic acid (KA)–induced seizures, Baran et al. performed a series of experiments with mannitol administration at different points in time following KA injection (1987). They discovered that mannitol infused during the period of time associated with cerebral edema attenuated status epilepticus and prevented the typical irreversible postepileptmic brain damage (if given within 10 min after the onset of generalized convulsion). Not only did the seizures stop after mannitol treatment, but noradrenaline levels measured 3 h after KA injection were within the control range. Mannitol infused before or after this period had little effect (1987).
Traynelis and Dingledine abolished 48 of 56 electrographic seizures in slices using media made 5, 15, or 30 mOsm hyperosmotic by agents restricted to the ECS, including mannitol, sucrose, raffinose, l-glucose, and dextran (1989). They noted that seizure suppression was seen in all media; however, seizures were abolished more quickly at higher mOsm concentrations. Membrane permeable compounds including glycerol and d-glucose were ineffective (1989).
A look at possible mechanisms
With the discovery that effects on voltage and ligand-gated ion channels were responsible for the anticonvulsant activity of many of our first-generation antiepileptics, synaptic theories of epileptiform synchronization and epileptogenesis have prevailed. Some diuretics appear to act by modulating synaptic activity. For example, acetazolamide blocks carbonic anhydrase, an enzyme that catalyzes the hydration and dehydration of CO2 into carbonic acid (i.e., bicarbonate and a proton). Although Cl− is the primary charge carrier through GABAA channels, bicarbonate also permeates the GABAA channel, causing the membrane response to be slightly more depolarizing than if only Cl− were carrying the charge. During intense synaptic activity, such as occurs during seizures, the Cl− currents through the GABAA channel lead to equilibration of the Cl− concentrations on either side of the membrane, so that the driving force for Cl− current is reduced (Huguenard & Alger, 1986; Misgeld et al., 1986). This is not the case for bicarbonate flux, which continues as long as carbonic anhydrase continues to catalyze the conversion of CO2 and water to and from bicarbonate and protons. The end result is a more pronounced depolarizing shift in the GABA response, which reduces the efficacy of GABAergic inhibition. Blocking extra- and/or intracellular carbonic anhydrase with acetazolamide or other inhibitors causes the bicarbonate concentrations to collapse along with the Cl− gradients, thereby blocking the depolarizing response (Staley et al., 1995; Staley & Proctor, 1999). Because anticonvulsants such as barbiturates and benzodiazepines prolong the GABA response, this effect of acetazolamide may be synergistic with the effects of these GABAergic anticonvulsants (Staley, 2004).
Nonsynaptic mechanisms may also be important in epileptiform synchronization. Taylor and Dudek demonstrated that synaptic transmission may not even be necessary for ictogenesis (1982), and similar findings were reported by Jefferys and Haas (1982). Using a low Ca+ with Mn2+ bath, chemically evoked postsynaptic potentials were blocked in slices. Electrical stimuli applied to the slices produced large-amplitude action potentials synchronized across a large CA1 population despite complete synaptic blockade. They hypothesized a combination of mechanisms including electrotonic coupling via gap junctions, electrical field or “ephaptic” effects, and fluctuations of extracellular ions. After nearly 30 years, these studies remain as compelling as they are difficult to interpret because of the multitude of effects of calcium on transmembrane ion channels and intracellular signaling cascades.
Other investigators demonstrated that neural activation can induce shrinkage of the ECS by 20% (Dietzel et al., 1982; Ransom et al., 1985). Ransom et al. found that neural stimulation resulted in increased [K+]o, resulting in ECS shrinkage that was blocked by Cl− free solution, SITS (4-acetamido-4-isothiocyanostilbene-2,2′-disulfonic acid), and furosemide. They suggested that a glial anion transport system was integral to activity-dependent ECS shrinkage. Dudek et al. later examined the effects of ECS size in the setting of total synaptic blockade and concluded that activity-dependent reduction of ECS and subsequent enhancement of nonsynaptic mechanisms of neuronal excitation may represent a positive feedback loop that contributes to the induction and maintenance of epileptogenesis (1990).
Uptake of K+ and Cl− in cultured astrocytes was blocked using various combinations of furosemide and bumetanide, and K+ uptake in the presence of ouabain was sensitive to the omission of Na+/Cl− medium (Kimelberg & Frangakis, 1985). This evidence supported the presence of an astrocytic K+ + Na+ + Cl− cotransport system. Osmotic manipulations confirmed astrocytic regulatory volume decreases after exposure to hypotonic medium, regulatory volume increases after subsequent exposure to isotonic medium, and osmotic shrinkage only in the presence of hypertonic medium. Kimelberg and Frangakis hypothesized that the activity-dependent ECS shrinkage, as seen in the rat optic nerve study, may be correlated with astrocyte swelling due to stimulation of cation plus Cl− cotransport systems (1985).
Shrinkage of the ECS is hypothesized to promote excitability, synchrony, and ultimately seizures (Lux et al., 1986; Traynelis & Dingledine, 1989). Initially ECS shrinkage can exaggerate the transient elevations of extracellular potassium associated with intense periods of neuronal activity, thereby increasing excitability. ECS shrinkage can also enhance electrical field effects that promote synchrony via two proposed mechanisms. As neighboring neurons move closer to one another they may adopt a similar refractory period during the momentary negative extracellular potential that occurs during action potentials. A synchronous firing of neurons will create a voltage gradient that results in electrical current preferentially traversing neighboring cells rather than ECS, as shrinkage of the ECS increases its electrical resistance. The current that does move through the ECS tends to depolarize, and, therefore, recruit silent neurons in the vicinity (Taylor & Dudek, 1984) Alternatively, the ECS voltage gradients can cause a transient redistribution of the major extracellular electrolytes (Na+ and Cl−) near activated neurons. Traynelis and Dingledine concluded that ECS shrinkage is a “critical determinant of the conditions that enable CA3 interictal input to precipitate an electrographic seizure in CA1” (1989).
The evidence for diuretics as antiepileptics
Hochman et al. examined the antiepileptic effects of high (millimolar) concentrations of the chloride cotransporter antagonist, furosemide, in a broad range of in vitro epilepsy models (1995). Monitoring ECS volume changes by measurements of the intrinsic optical signal, a measure of light scattering by brain tissue (Lipton, 1973), MacVicar and Hochman blocked stimulus evoked changes in light transmission through the CA1 region of hippocampal slices with furosemide (1991). Building on this work, Hochman et al. used direct electrical stimulation of the Shaffer collaterals to demonstrate furosemide-blockage of afterdischarge activity as well as associated optical changes, which were reversed after furosemide washout (1995). In addition to blocking epileptiform activity, furosemide appeared to dissociate synchronized activity (spontaneous discharges decreased) and excitability (increased amplitude of evoked epileptiform discharges). Furosemide successfully blocked several different physiologic models of epilepsy: hyperkalemic bath (model of hyperexcitability), hypomagnesemic bath (anticonvulsant resistant model), 0 Ca2+ bath (synaptic blockade), 4-AP (K+ channel antagonist), bicuculline (GABAA antagonist), and kainic acid (status epilepticus and kindling model). They concluded that furosemide must act at a common mechanism necessary for the generation of synchronized bursting, and based on their optical signal data, they proposed that furosemide’s action was related to cell volume regulation due to glial swelling. Gutschmidt et al. confirmed similar findings, demonstrating furosemide blockade of seizures in slices bathed in solutions containing low Ca2+, low Mg2+, and 4-AP (1999).
Hochman et al. further tested the hypothesis that the antiepileptic effects of furosemide are mediated through actions on Cl transport by mimicking its anticonvulsant effect with low extracellular Cl− (1999). Epileptiform discharges in slices perfused with high K+, bicuculline, or 4-AP were blocked by perfusion of low- Cl− media. Furosemide causes a depolarizing shift in EGABA by blocking K+ and Cl− cotransport, allowing equilibration between intra- and extracellular chloride concentrations, whereas low [Cl−]o reduces inhibitory postsynaptic potential driving force by directly diminishing the transmembrane chloride gradient. Both conditions reduce the driving force for inward Cl− flux, which diminishes the efficacy of GABAergic inhibition, resulting in stimulus-evoked hyperexcitability, as evidenced by evoked epileptiform bursts of increased magnitude. Because they demonstrated nearly identical effects on neuronal hypersynchronization, furosemide and low [Cl−] o were felt to exhibit their antiepileptic properties on glial Na+/K+/2Cl− cotransport. They also note that their data did not depend on the actual location of the Na+/K+/2Cl− cotransporter and acknowledged the possibility of a neuronal Na+/K+/2Cl− cotransporter (1999). Unresolved puzzles are the prolonged time required for the anticonvulsant effect of low Cl− solutions (about an hour), and the very high concentration of furosemide needed for the profound anticonvulsant effect: Hochman et al. used 2.5, about 25 times the concentration needed to completely block potassium chloride cotransporter 2 (KCC2) and sodium potassium chloride cotransporter 1 (NKCC1) (Gillen et al., 1996) and five times the amount needed to block other potassium-chloride cotransporters (Gagnon et al., 2007). Without suppressing normal neuronal excitability, Hochman and Schwartzkroin confirmed that the antiepileptic effects of chloride cotransport antagonism are due to desynchronization (2000).
With their accumulated techniques and experience, Haglund and Hochman investigated the role of ECS modulation in human susceptibility to ictal events (2005). In adult patients undergoing resection of an epileptic focus (still on their baseline antiepileptic medications) investigators intraoperatively measured intrinsic optical signal and electrocorticography following a single intravenous bolus of furosemide (20 mg). Within 10–30 min after administration, the frequency of interictal spikes was reduced on average by 60% in the five patients tested. In eight patients, they examined furosemide-induced resistance to direct cortical electrical stimulation and reported complete blockade in 4, and >50% reduction of afterdischarge duration and amplitude in the remainder. If furosemide raised epileptic threshold through modulation of the ECS, it would be expected that osmotic agents would also have similar effects. They repeated the above studies in four patients with a single 50-g intravenous injection of mannitol and found the same 60% reduction of spontaneous spiking frequency and 42% reduction of afterdischarge duration following direct cortical stimulation. They concluded that (1) furosemide and mannitol suppressed highly resistant epileptic activity, (2) the effect was seen in all patients with both mesial temporal and neocortical epilepsy, and (3) concomitant EEG activity from nonepileptic sites was preserved, suggesting that epileptic suppression was not due to global suppression of neuronal activity (2005). The means by which mannitol is administered may alter its affect, as others have found that boluses of mannitol alter the blood–brain barrier and may provoke seizures (Marchi et al., 2007).
These data support the hypothesis that diuretics inhibit epileptic activity by inducing volume shifts. This might reflect an expansion of the extracellular space and concomitant reduction in ephaptic interactions, but there are many other potential mechanisms. For example, alterations in cellular volume trigger a cascade of intracellular signals designed in part to activate mechanisms of volume restoration (Kahle et al., 2006, 2008; Hoffmann et al., 2009), but the anticonvulsant effects of activating these signaling cascades are not known. Additional mechanisms of action may also be implicated through inhibition of the K+-Cl cotransporter KCC2 (Gutschmidt et al., 1999; Viitanen et al., 2010) and in their effect on chloride uptake mediated by NKCC1 at the axon initial segment (Khirug et al., 2008), or via inhibition of Cl/HCO3 exchange (Gutschmidt et al., 1999), although these mechanisms do not address the high concentrations of furosemide required in vitro (but perhaps not in vivo: Haglund & Hochman, 2005) or the curious time dependence of the low-chloride media. It has also been suggested that chloride-dependent steps in glutamate transport may be responsible for these anticonvulsant effects (Staley, 2002).
In addition to their broad safety profile, the attractiveness of diuretics as antiepileptics lies both in their broad efficacy in in vitro and in vivo models of epilepsy as well as the selectivity of their effects on epileptic versus normal activity, which unfortunately is not shared by most of our current antiepileptic medications.