The mechanism by which anesthetics block function remains incompletely understood. The first modern theory was proposed by Hans Meyer and again by Charles Overton [2, 3]. According to that theory, anesthetics diffuse into the lipid bilayer of cell membranes. This changes the cell’s chemistry by affecting the solubility of ions and other molecules in fatty substances. Meyer tested the solubility of various molecules and ions in olive oil, which was meant to represent fatty molecules in brain cells. He compared olive oil’s solubility with and without anesthetics added. He then tested the anesthetics on tadpoles to observe any behavioral effects. While his tadpole experiments were imprecise and subjective, Meyer was able to correlate lipid solubility with anesthetic concentration [2]. Overton conducted experiments similar to Meyer’s and was also able to correlate lipid solubility with the concentration of anesthetic [3].

Similar ideas followed those original proposals. For example, Miller and colleagues proposed that anesthetic accumulation in the membrane bilayer reduced the effectiveness of channel proteins which in turn prevented proper ion flow [4]. However, other investigators raised objections: Drugs that are soluble in lipids would be expected to have a similar effect, but do not. Hydrophobic molecules such as halogenated alkanes fail to induce narcosis [5]. Temperature increases would also be expected to increase membrane fluidity and thus increase the anesthetic effect, but this effect is not seen [6]. Nevertheless, most investigators today consider that anesthetics act on membrane proteins, channels, or receptors, with current theories emphasizing membrane lateral pressures, and the models of protein binding originally introduced by Franks and Lieb [6].

Linus Pauling proposed an entirely different idea much earlier: that anesthetics stabilized hydrate structures, preventing normal electrical signals and ion flow and thereby inducing narcosis [7]. Pauling’s idea lacked direct evidence, and was apparently not followed up.

Recently, it has become clear that cells contain considerable amounts of interfacial water [8]. Most water molecules lie within one nanometer of some hydrophilic surface; therefore, cell water is mainly interfacial water. Studies of interfacial water—now termed “exclusion zone” (EZ) water because of its profound solute-exclusionary nature—have shown that EZ water builds extensively next to hydrophilic surfaces, including gels and biological surfaces [8]. Given the abundance of EZ water in the cell, the question arose whether EZ water might be a possible target of anesthetic action.

Recent evidence has given force to this hypothesis. A threshold amount of water needs to be present near hydrophilic protein sites in order for anesthetics to work effectively [9]. It is reasonable then to think that anesthetics might interact in some way with water to produce narcosis.

One particular hydrophilic material that creates a prominent exclusion zone is the polymer Nafion. Nafion has a Teflon backbone with many anionic sulfonate groups attached, so it resembles a cellular membrane with a negative charge. It can thus bond to water. Nafion has been used in many studies of EZ properties, and was used as a model for the current experiments.

The aim of this work was to determine if there is a relationship between water’s EZ and anesthesia’s mechanism of producing narcosis. The results confirmed this expectation.

All concentrations of local anesthetics affected the exclusion zone significantly. This was true of both Lidocaine (Fig 3) and Bupivacaine (Fig 4). In general, EZ size diminished with higher anesthetic concentrations.

A surprising result occurred at the lower concentrations of both Lidocaine and Bupivacaine. At 0.34 mM Lidocaine and 0.07 mM Bupivacaine, the exclusion zone expanded substantially. While the standard deviation of this value was relatively high, the standard deviation divided by the mean value was similar to other values (Tables 1 and 2). Hence, the trials were fairly consistent over the full range of concentrations.

Much like the local anesthetics, the general anesthetic Isoflurane increased EZ size at low concentration (Fig 5 and Table 3). When Isoflurane concentration was above 0.1% (v/v), however, EZ size diminished significantly and continued to diminish with further concentration increase. By 0.3% the EZ diminished to one third of its initial size.

Fig 6 shows the reversibility of Bupivacaine’s effect. Once Bupivacaine was added, the exclusion zone diminished in size. After the microsphere solution was replaced with a control microsphere solution that contained no anesthetic, the exclusion zone returned to its original size, i.e., the size before the anesthesia was added. Similar results were obtained with Isoflurane.

The results show that anesthetics in clinically relevant concentrations have profound effects on interfacial water. We studied two local anesthetics, Lidocaine and Bupivacaine, as well as an inhalational anesthetic Isoflurane. All three anesthetics showed similar effects: at an extremely low concentration they expanded the zone of interfacial water (EZ). At higher concentrations they diminished EZ size in a concentration-dependent manner. The effects were fully reversible.

These effects follow common clinical experience. The extremely low anesthetic doses that patients experience during induction produce excitatory effects (known as Stage II or the “excitement” stage), while higher concentrations produce increasingly stronger levels of narcosis (known as Stages III and IV or the “surgical” and “overdose” stages). The question arises how these effects on interfacial water might help explain narcosis.

One possible explanation derives from the fact that cells are filled with interfacial water [13]. This (EZ) water bears negative charge [8]. Thus, at least some of the cell’s negative charge and negative electrical potential arise from EZ water. That electrical potential is evidently crucial for action potential generation, which bears responsibility for cellular communication and other cellular functions. If this intracellular potential is diminished through diminution of EZ water, then the cell becomes unable to properly function, and particularly unable to communicate. Proteins cannot fold, and currents cannot flow through membranes.

This result might help explain the observation that anesthetic-like inhibitory action occurs not only in animals with nerves but also in simple organisms and even single-celled creatures. In experiments conducted by Keil et al. [14], five different anesthetics were all found to inhibit respiration and meiosis in yeast cells, which lack nervous systems altogether. Similarly, when nematodes, which have very simple nervous systems, are exposed to anesthetics, the nematodes are initially “excited” and race around their environment until full narcosis sets in and they become unresponsive to external stimuli such as needle prods [15]. These experiments indicate that anesthetic-like effects extend to not just the advanced nervous systems of larger animals, but also to general biological functions that exist in more primitive species. The excitatory response at low dose is confirmed as well.

Anesthesia’s ability to affect the EZ’s size in cells can also explain how narcosis can be reversed by high pressure. Miller et al. describe how elevated atmospheric pressures antagonize the effects of both liquid and gaseous anesthetics [16]. It would seem that if diminished EZ caused narcosis, then increased pressure would need to increase the size of the EZ to weaken the anesthetic effect. Ypma and Pollack demonstrated that elevated pressure does in fact significantly increase the size of the EZ [17]. This result adds strength to the hypothesized relationship between narcosis and cellular water.

Thus, anesthetic action is a rather general effect that occurs widely across the phylogenetic spectrum. This generality squares with the results found here. Interfacial water is the most common substance in all cells. Water constitutes two thirds of cell volume; however, the water molecule is so small that numerous molecules are required to make up that two-thirds fraction. Thus, more than 99% of the cell’s molecules are water molecules. Hence, the most common molecule in the cell appears to be a common target of anesthetics. This finding does not preclude other sites of anesthetic action such as those generally accepted; however, it goes some way toward explaining the generality of the anesthetic effect.