Inhibition of KCC2 in mouse spinal cord neurons leads to hypersensitivity to thermal stimulation

KCC2, a neuronal-specific K-Cl cotransporter, is involved in pain perception physiology through its effects on postsynaptic inhibition in spinal cord neurons. We injected a newly identified, highly potent and selective inhibitor of KCC2 (D4), an inactive structural variant (D4.14), and the Na-K-2Cl cotransporter (NKCC1) inhibitor, bumetanide, into the intrathecal space of mice to measure their effect on heat-evoked nociceptive responses.

Commercially available intrathecal catheters were modified and surgically placed into 2 cohorts of 10 mice. After recovery from the procedure, the mice were injected with D4, D4.14, and bumetanide through this catheter. Nociceptive measurements (hotplate assay, tail flick assay) were performed after injection of each of the test drugs and compared with vehicle controls.

Two mice in each cohort were omitted because of postprocedure complications. There was a statistically significant decrease (P < 0.01) in withdrawal latency after injection of the active KCC2 inhibitor but not after injection of the inactive compound (P = 0.78), as measured by hotplate assay at 55°C. Injection of bumetanide significantly increased withdrawal latency (P = 0.02) at the same temperature. These results were confirmed using tail flick assays performed at 49°C.

Inhibition of KCC2 by D4 led to decreased heat-evoked withdrawal latency in mice, as measured by hotplate and tail flick assays, whereas inhibition of NKCC1 by bumetanide resulted in increased response latencies to heat stimuli as measured by both of these nociceptive tests.