Synergistic depression of NMDA receptor-mediated transmission by ketamine, ketoprofen and L-NAME combinations in neonatal rat spinal cords in vitro

Abstract

Background and purpose: Spinal N-methyl-D-aspartate (NMDA) receptor/cyclooxygenase (COX) and nitric oxide synthase (NOS) pathways play a major role in nociceptive processing, and influencing them simultaneously may induce synergistic analgesia. This study determined the spinal antinociceptive interactions between ketamine (NMDA receptor channel blocker), ketoprofen (COX inhibitor) and L-NAME (NOS inhibitor) combinations.

Experimental approach: Using an in vitro neonatal rat spinal cord preparation, two A-fibre-mediated reflexes, the monosynaptic reflex (MSR) and the low-intensity excitatory postsynaptic potential (epsp), and one C-fibre-mediated reflex, the high-intensity epsp, were evoked electrically. The effect of drugs and drug combinations on these reflexes was assessed and the type of interaction determined by isobolographic analysis.

Key results: Infusion of ketamine alone decreased all three reflexes. That of ketoprofen decreased both the low and the high-intensity epsp only. Infusion of L-NAME alone produced no significant effects. Co-infusion of fixed ratios of IC(40) fractions of both (ketamine+ketoprofen) and (ketamine+L-NAME) were synergistic for depressing the low and the high-intensity epsps. The interaction was sub-additive for both combinations on the MSR. The only significant effect for the (ketoprofen+L-NAME) combination was synergism on the high-intensity epsp.

Conclusions and implications: All three combinations synergistically depressed nociceptive spinal transmission, and both ketamine and ketoprofen and ketamine and L-NAME combinations did so with potentially decreased motor side effects. If such combination profiles also occur in vivo, the present findings raise the possibility of ultimate therapeutic exploitation of increased analgesia with fewer side effects.

Figure 1

Effects of ketamine+ketoprofen combinations on the synaptic responses. The combinations depressed the low-intensity epsp (b) and the high-intensity epsp (c), but had a minor effect on the MSR (a). Recordings in the left panels show the control responses (time 0 min), each one from a different preparation, those in the middle panels represent the depressive effects of the ketamine+ketoprofen combinations (time 160 min) and those in the right panels illustrate their recovery after 90 min of drug-free medium (time 250 min). (d) Time course showing the actions of ketamine+ketoprofen combinations, each concentration infused for 35 min, on the different components of the synaptic response. Fractions were 0.045:0.955 for the MSR and the low-intensity epsps, and 0.048:0.952 for the high-intensity epsp. Each point represents the mean±s.e.mean of four preparations for each one of the synaptic responses. epsp, excitatory postsynaptic potential; MSR, monosynaptic reflex.

Figure 2

Percent maximal possible effect (MPE%) of ketamine, ketoprofen and L-NAME alone on the MSR (a), the low-intensity epsp (c) and the high-intensity epsp (e), MPE% for the actual combinations of ketamine:ketoprofen, ketamine:L-NAME and ketoprofen:L-NAME on the MSR (b), the low-intensity epsp (d) and the high-intensity epsp (f). Each point represents the mean±s.e.mean of four preparations for each one of the synaptic responses, except for ketamine and ketoprofen alone and ketoprofen+L-NAME combination on the low-intensity epsp, for which five preparations per treatment were used. epsp, excitatory postsynaptic potential; L-NAME, N^ω-nitro-L-arginine methyl ester; MSR, monosynaptic reflex.

Figure 3

Isobolograms for the MSR (a), the low-intensity epsp (c) and the high-intensity epsp (d, e) at an effect level of 40% depression of the maximal possible effect for the combinations ketamine+ketoprofen in fixed-ratio proportions (0.045:0.955 for the MSR and the low-intensity epsp, and 0.048:0.952 for the high-intensity epsp). Points A represent the calculated additivity quantities for these proportional combinations. Points C are the combination points determined experimentally with these same proportional mixes. Confidence intervals for point C in (a) are too wide and hence not plotted. Percent maximal possible effect (MPE%) of ketoprofen alone, ketamine alone and their composite additive line in the above cited proportions are shown for the low-intensity epsp (b) and the high-intensity epsp (e). epsp, excitatory postsynaptic potential; MSR, monosynaptic reflex.

Figure 4

Isobolograms for the MSR (a), the low-intensity epsp (b) and the high-intensity epsp (c) at an effect level of 40% depression of the maximal possible effect for the combination ketamine+L-NAME in a fixed-ratio proportion (0.8805:0.1195 for all three responses). Points A represent the calculated additivity quantities for these proportional combinations. Points C are the combination points determined experimentally with these same proportional mixes. epsp, excitatory postsynaptic potential; L-NAME, N^ω-nitro-L-arginine methyl ester; MSR, monosynaptic reflex.

Figure 5

Isobolograms for the low-intensity epsp (a) and the high-intensity epsp (b) at an effect level of 40% depression of the maximal possible effect for the combination ketoprofen+L-NAME in a fixed-ratio proportion (0.8805:0.1195 for both responses). Points A represent the calculated additivity quantities for these proportional combinations. Points C are the combination points determined experimentally with these same proportional mixes. epsp, excitatory postsynaptic potential; L-NAME, N^ω-nitro-L-arginine methyl ester.