Ketamine potentiates cerebrocortical damage induced by the common anaesthetic agent nitrous oxide in adult rats

Abstract

For general anaesthesia, patients usually receive a combination of drugs, all of which are classified as gamma-amino-butyric acid (GABA) agonists, with two notable exceptions - ketamine and nitrous oxide (laughing gas, N(2)O) - which are antagonists of N-methyl-D-aspartate (NMDA) glutamate receptors. At clinically relevant doses both ketamine and N(2)O, like other NMDA antagonists, have the potential to induce psychotomimetic reactions in humans and to cause pathomorphological changes in cerebrocortical neurons in rat brain. Because drug combinations used in clinical anaesthesia sometimes include both ketamine and N(2)O, we undertook experiments to evaluate whether augmented neurotoxicity results from their combined use. Ketamine and N(2)O were administered alone or in combination by various dosing regimens to adult female rats for a duration of 3 h and the severity of cerebrocortical neurotoxic changes was quantified histologically. Because GABA agonists are known to protect against the psychotomimetic and neurotoxic effects of NMDA antagonists, we also evaluated whether the combined neurotoxicity of ketamine+N(2)O can be prevented by certain commonly used GABA agonists. When ketamine and N(2)O were used in combination the neurotoxic reaction was enhanced to a degree much greater than can be explained by simple additivity. The apparent synergistic interaction was particularly striking when low doses of the agents were combined, the degree of toxic synergism at higher doses being masked by a ceiling effect. GABA agonists protected against ketamine/N(2)O neurotoxicity. It is recommended that this information be taken into consideration in the selection of drugs to be used in multi-agent protocols for general anaesthesia.

Figure 1

Neurotoxicity in PC/RSC neurons caused by exposure of adult rats for 3 h to either N₂O or ketamine is dose-dependent. Dose-dependent curves are steep and the calculated EC₅₀ for N₂O is 118 vol% and calculated ED₅₀ for ketamine is 47.5 mg kg⁻¹ i.p. The control condition for N₂O experiments is normobaric or hyperbaric air or for ketamine experiments is saline i.p. and is depicted as 0 vol% and 0 mg kg⁻¹, respectively (n=⩾7 per treatment group).

Figure 2

N₂O significantly potentiates the neurotoxicity caused by ketamine. Adult female rats were exposed to ketamine either alone or in combination with various concentrations of N₂O (as indicated) for 3 h. The ketamine neurotoxicity curve is shifted markedly upward and to the left by combination with concentrations of N₂O (50 or 75 vol%) which by themselves are not neurotoxic. The number of vacuolated neurons in PC/RSC in the presence of N₂O+ketamine is significantly higher than in the presence of the corresponding dose of ketamine alone (P<0.05). The control condition for N₂O experiments is normobaric of hyperbaric air or for ketamine experiments is saline i.p. and is depicted as 0 vol% and 0 mg kg⁻¹, respectively (n=10–12 per treatment group).

Figure 3

Ketamine significantly potentiates the neurotoxicity caused by N₂O. Adult female rats were exposed to N₂O either alone or in combination with various doses of ketamine (as indicated) for 3 h. Potentiation of N₂O neurotoxicity is particularly marked when a non-toxic concentration of N₂O is combined with a low dose of ketamine which by itself is either non-toxic (20 mg kg⁻¹) or only slightly toxic (40 mg kg⁻¹). The number of vacuolated neurons in PC/RSC in the presence of ketamine+N₂O is significantly higher than in the presence of the corresponding concentration of N₂O alone (P<0.05). The control condition for N₂O experiments is normobaric or hyperbaric air or for ketamine experiments is saline i.p. and is depicted as 0 vol% and 0 mg kg⁻¹, respectively (n=10–12 per treatment group).

Figure 4

Isobolographic curve for the interaction between N₂O and ketamine. The EC₅₀ for N₂O is plotted on the Y axis and the ED₅₀ for ketamine on the X axis. The line connecting these two points is the theoretical line of additivity. Point A is the theoretical additive ED₅₀ point. The experimental ED₅₀ point (B) for the combination falls below the theoretical line of additivity suggesting a synergistic (supra-additive) interaction.

Figure 5

Either inhalational anaesthetics (halothane and isoflurane) or intravenous anaesthetics (pentobarbital and diazepam) successfully block the neurotoxic reaction caused by N₂O and ketamine. (^*)=P<0.0005 for comparison of D, E, F or G with C; (^*)=P<0.0005 for comparison of A or B with C. The P values for N₂O+ketamine as a treatment condition by itself vs N₂O+ketamine in combination with a GABAergic agent exceeded Bonferroni corrected (P=0.008) levels.

Figure 6

Histological appearance of the PC/RSC 3 h after treatment with: (A) Ketamine alone (40 mg kg⁻¹ i.p.). There is a very mild toxic reaction evidenced by vacuolization of a single neuron (arrow); (B) N₂O alone (75 vol%). There is no detectable toxic reaction; (C) N₂O (75 vol%)+ketamine (40 mg·kg⁻¹ i.p.). There is a severe toxic reaction evidenced by conspicuous vacuoles in all neurons in the field (multiple arrows); (D) N₂O (75 vol%)+ketamine (40 mg kg⁻¹+isoflurane (1.5 vol%). PC/RSC neurons appear normal. Isoflurane has completely blocked the neurotoxic reaction that would otherwise be caused by N₂O+ketamine. (E) Control condition – exposed only to normobaric air. PC/RSC neurons have a normal appearance (magnification all panels ×480).