Generation of Na_v1.8Cre mice. (A) Diagram of the native Na_v1.8 allele, Na_v1.8Cre targeting construct, and Na_v1.8Cre-targeted allele before and after excision of neo^r. (B and C) Histological localization of Na_v1.8Cre-mediated β-galactosidase activity. Serial 10-μm sections from 4% paraformaldehydefixed Na_v1.8Cre^+/-,R26R^+/- animals were stained with antibodies to β-galactosidase (B) and with anti-Na_v1.8 polyclonal antisera (C). Arrows highlight double-labeled cells. (Scale bars, 50 μm.) (D) Southern blot with BamHI and external probe confirms correct targeting and excision of neo^r. The 6.5-kb WT band is seen in all lanes, and the 8.5- and 7.3-kb bands represent the targeted allele before (+neo^r) and after (-neo^r) excision of neo^r, respectively.

Generation and analysis of Na_v1.7 floxed mice. (A) Structure of the native Na_v1.7 allele, Na_v1.7 targeting construct, fNa_v1.7 allele, fNa_v1.7 allele after excision of neo^r, and Na_v1.7 knockout allele. (B) Southern blotting with EcoRI and the external probe confirms correct targeting. (C) Southern blotting with ApaI and internal probe confirms the removal of neo^r cassette. (D) Southern blotting confirms the deletion of exons 14 and 15 in Na_v1.7^-/-. (E) PCR was used to detect exon 14-15 deletion in genomic and cDNA from DRG but not spinal cord in Na_v1.7R^-/-.

Electrophysiology of Na_v1.7^-/-.(A) TTX-sensitive and -resistant peak sodium current density in Na_v1.7R^-/- (black) (n = 7) and fNa_v1.7 (white) (n = 14) mice. (B) TTX-resistant currents in WT (hatched) (n = 22), Na_v1.8Cre heterozygotes (filled) (n = 22), and homozygous (n = 26) mice. (C-F) Responses of lamina V dorsal horn neurons (L3 and L4) receiving input from the hindpaw to peripheral stimuli in Na_v1.7 R^-/- (black) (n = 14) and littermate fNa_v1.7 (white) (n = 18) mice. (C) Evoked responses to von Frey hairs showed a mechanical deficit in Na_v1.7R^-/- mice (P = 0.048). Temperature (water jet) (D) and pinch, brush, and noxious cold (E) applied to the peripheral receptive field over 10 s were normal. Data are expressed as mean spikes (±SEM) evoked over 10 s. (F) Response to transcutaneous electrical stimulation of the receptive field (train of 16, 2-ms-wide electrical pulses, at 0.5 Hz, at three times C-fiber threshold). Spikes evoked between 0 and 50 ms were classified as caused by A-fiber input, and those evoked between 50 and 250 ms were classified as caused by C-fiber input. “Input” is the number of spikes evoked by the first stimulus of the train (50-800 ms) and reflects nonpotentiated C-fiber-evoked dorsal horn neuron response. Excess spikes, additional spikes recorded above the predicted constant baseline response, are a measure of “wind-up,” defined as increased neuronal excitability to repeated constant stimulation.

Acute pain behavior of Na_v1.7R^-/- mice. (A) Noxious thermal stimulation of fNa_v1.7 (white) and Na_v1.7R^-/- (black) mice by using Hargreave's apparatus. The latency of hindpaw withdrawal was significantly increased in Na_v1.7R^-/- mice (11.01 ± 0.47, n = 14, and 8.23 ± 0.67, n = 12, respectively) (P = 0.003, t test). (B) Response to noxious thermal stimulation by using the hotplate apparatus was not significantly different in fNa_v1.7 (n = 12) and Na_v1.7R^-/- (n = 17) mice (P = 0.27, 0.11, and 0.79, t test). (C)Na_v1.7R^-/- mice (396.4 ± 4.0, n = 19) showed profound analgesia to noxious mechanical pressure when using the Randall-Selitto apparatus compared with fNa_v1.7 mice (195.5 ± 4.8, n = 18) (P < 0.0001, t test). (D) Response to mechanical stimulation when using von Frey hairs was not significantly different in fNa_v1.7 (n = 11) and Na_v1.7R^-/- (n = 19) mice.

Inflammatory pain in Na_v1.7R^-/-. (A) Analysis of behavior of fNa_v1.7 (white) and Na_v1.7R^-/- (black) mice in models of inflammatory pain. (Aa) Pain behavior of fNa_v1.7 (n = 14) and Na_v1.7R^-/- (n = 17) mice after intraplantar injection of 20 μl of 5% formalin. Time spent licking/biting the injected hindpaw in phase I (1-10 min) and phase II (10-55 min) was recorded. There is a reduction in phase I (P = 0.03, t test) (fNa_v1.7, 93.1 ± 7.2; Na_v1.7R^-/-, 70.4 ± 6.6). In phase II, Na_v1.7R^-/- mice also showed a significant reduction in pain behavior (P = 0.004, t test) (101.3 ± 18.9 and 220.9 ± 34.8 respectively). (Ab) Time course of the formalin response (P < 0.05 at 5-, 20-, 25-, and 30-min time points). (Ac) Thermal hyperalgesia after intraplantar injection of 20 μl of CFA. fNa_v1.7 mice developed pronounced hyperalgesia (0.56 ± 0.1, n = 9) from day 1, whereas Na_v1.7R^-/- mice did not (0.97 ± 0.06, n = 14) (P < 0.001, ANOVA; P < 0.05 at all time points). (Ad) Mechanical allodynia induced by intraplantar injection of 20 μl of CFA. fNa_v1.7 mice developed pronounced allodynia from day 1 (0.50 ± 0.16, n = 8), whereas the Na_v1.7R^-/- mice did not (1.05 ± 0.12, n = 13) (P = 0.012, ANOVA; P < 0.05 at days 2, 3, 4, and 10). (Ae) Thermal hyperalgesia after intraplantar injection of 20 μl of 2% carrageenan. fNa_v1.7 mice developed pronounced hyperalgesia (0.47 ± 0.03, n = 7) within an hour, whereas Na_v1.7R^-/- mice did not (1.07 ± 0.04, n = 5) (P < 0.001, ANOVA; P < 0.05 at all time points). (Af) Thermal hyperalgesia after intraplantar injection of 500 ng of NGF. fNa_v1.7 mice (n = 15) developed thermal hyperalgesia in a biphasic pattern, whereas Na_v1.7R^-/- mice (n = 8) showed no phase I (first hour) and a reduced phase II (P < 0.05 at 0.25, 0.5, 1, 2, 6.5, and 8 h, ANOVA). (B) Inflammatory pain is normal in Na_v1.8Cre mice. Analysis of behavior of Na_v1.8Cre^+/- (black) and C57BL/6 (white) mice in models of inflammatory pain. (Ba) Behavior of Na_v1.8Cre^+/- (n = 6) and C57BL/6 (n = 6) mice after intraplantar injection of 20 μl of 5% formalin was not different between groups in phase I (107 ± 12.6 s and 96.1 ± 10.3 s, respectively; P = 0.51) and phase II (230 ± 47.7 and 290 ± 45.4 s, respectively; P = 0.38, t test). (Bb) Thermal hyperalgesia after intraplantar injection of 20 μl of CFA was not different in Na_v1.8Cre^+/- (0.58 ± 0.05, n = 6) and WT (0.55 ± 0.05, n = 6) littermates (P = 0.79, ANOVA). (Bc) Mechanical allodynia after intraplantar injection of 20 μl of CFA was not different in Na_v1.8Cre^+/- (0.30 ± 0.16, n = 6) and WT (0.27 ± 0.04, n = 6) littermates (P = 0.74, ANOVA). (Bd) Thermal hyperalgesia induced by intraplantar injection of 20 μl of 2% carrageenan was not different in Na_v1.8Cre^+/- (0.40 ± 0.26, n = 9) and C57BL/6 (0.36 ± 0.03, n = 7) mice (P = 0.31, two-way ANOVA). (Be) Thermal hyperalgesia induced by intraplantar injection of 500 ng of NGF was not different in Na_v1.8Cre^+/- (0.58 ± 0.02) (n = 6) and C57BL/6 (0.56 ± 0.03) (n = 6) mice (P = 0.83, two-way ANOVA).