Fig. 4: Kenpaullone re-normalizes EGABA in spinal cord dorsal horn by increasing Kcc2 expression/KCC2 function in mice with neuropathic pain.
a Top left: Laminae I–II area of spinal cord dorsal horn (SCDH) is highlighted (yellow). Top right: image representation of laminae I–II area before and after laser capture microdissection. Bottom, bar diagram: KP (10 mg/kg) rescues significantly attenuated Kcc2 mRNA expression in the SCDH after nerve constriction injury (PSNL) vs. vehicle-treated mice; KP injected daily for 7 days after PSNL, SCDH microdissected 16 h after last injection of KP. Data are represented as mean values ± SEM. n = 4 mice/group, ***p = 0.0002, ****p < 0.0001 one-way ANOVA. b KP treatment increases KCC2 expression in the SCDH. Left-hand micrographs: representative KCC2 immuno-staining ipsilateral vs. contralateral to injury (see antibody validation in Supplementary Fig. 2a) of the SCDH in nerve constriction injury (PSNL), region-of-interest for densitometric measurement of KCC2 in the SCDH outlined with dotted white line in upper-left micrograph, focus on Rexed layers I–II because of their relevance for neurotransmission of nociceptive afferent signals. KCC2 signals were normalized for Nissl stain (green fluorescent signal, inlet micrographs, see Supplementary Fig. 4a), which did not differ between conditions and sides, see Supplementary Fig. 4a, b. Scale bar in upper left micrograph and inlet = 100 µm, valid for all panels. Bar diagrams: KP (10 mg/kg; daily treatment for 7 days after PSNL) increases KCC2 protein expression in SCDH vs. vehicle control in PSNL nerve constriction, ipsilateral to injury, no difference in KCC2 signal contralaterally. Data are represented as mean values ± SEM. n = 6 mice/vehicle control group, n = 7 mice/KP group, *p = 0.029; **p = 0.0026, one-way ANOVA. c Mechanical hypersensitivity in juvenile mice (5–7 weeks of age) after PSNL (grey bars) and its complete behavioral recovery after early intervention with KP (30 mg/kg; pink bars; 1× injection pre-PSNL, every 24 h post-PSNL). Data are represented as mean values ± SEM. n = 5 mice/group for all groups, behavioral assessment 72 h after KP injection. Note accentuated responses for sensitization and rescue in younger mice when comparing with adult mice as shown in Fig. 3a. ***p = 0.0005 one-way ANOVA. d Perforated patch-clamp recordings showing Cl−-extrusion capacity (EGABA) in lamina-II neurons from spinal cord slices of the same juvenile mice as treated in panel c, EGABA measured using the perforated patch method, illustrated by the schematic, 1–3 neurons per mouse. Left: representative I–V plot. Right: bar diagram showing quantification of EGABA indicating a significant depolarizing shift in sham vs. PSNL with vehicle treatment (“PSNL”). Note significant hyperpolarization in response to KP treatment in PSNL mice. Of note, PSNL plus KP was not different from sham injury. Data are represented as mean values ± SEM. n = 9 neurons (sham), n = 10 neurons (PSNL), n = 12 neurons (PSNL + KP); ****p < 0.0001, ***p = 0.0004, one-way ANOVA. Source data are provided as source data files.
