Pain without nociceptors? Nav1.7-independent pain mechanisms

Abstract

Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.

Graphical abstract

Figure 1

Comparison of Transgenic Mice Reveals Tissue-Specific Roles for Nav1.7 in Mechanical and Cold Allodynia after CCI Surgery as Well as a Role Specifically in Sympathetic Neurons after SNT Surgery Behavioral responses of different Nav1.7 tissue-specific knockouts to the von Frey and acetone test following CCI (A) and L5 SNT surgery (B). Nav1.7^Nav1.8 (blue squares, n = 9) do not develop CCI-induced cold allodynia (Aa) but do develop mechanical allodynia (Ab) in comparison to littermate mice (white squares, n = 8). Nav1.7^Advill (red squares, n = 8) do not develop CCI-induced cold (Ac) or mechanical allodynia (Ad) in comparison to littermate mice (white squares, n = 9). Nav1.7^Wnt1 (green squares, n = 9) do not develop CCI-induced cold (Ae) nor mechanical allodynia (Af) in comparison to littermate mice (white squares, n = 6). Nav1.7^Nav1.8 (blue squares, n = 6) develop both SNT-induced cold (Ba) and mechanical allodynia (Bb) in comparison to littermate mice (white squares, n = 6). Nav1.7^Advill (red squares, n = 6) develop both SNT-induced cold (Bc) and mechanical allodynia (Bd) in comparison to littermate mice (white squares, n = 9). Nav1.7^Wnt1 (green squares, n = 9) do not develop SNT-induced cold allodynia (Be) in comparison to littermate mice (white squares, n = 12). Data analyzed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean ± SEM. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (individual points). See also Figure S1.

Figure 2

Spinal Nerve Transection Fails to Trigger Sympathetic Sprouting in Nav1.7^Wnt1 Mice, which Can Be Sensitized by Norepinephrine (A) Yellow arrows show examples of sympathetic sprouting (tyrosine hydroxylase, red) into the ipsilateral DRG following SNT surgery (scale bar = 100 μm). (B) An example of a contralateral DRG showing no sympathetic sprouting (tyrosine hydroxylase, red) following SNT surgery (scale bar = 200 μm). (C) An example of a sympathetic “basket” (tyrosine hydroxylase, red) formed around a large diameter (N52, green) DRG neuron (scale bar = 20 μm). (D) Quantitation of sympathetic sprouting into the ipsilateral and contralateral L4 DRG following SNT. Littermates (white columns, n = 3), Nav1.7^Nav1.8 (blue columns, n = 3), Nav1.7^Advill (red columns, n = 3), and Nav1.7^Wnt1 (green columns, n = 3). (E) Quantitation of sympathetic sprouting into the L4 DRG following SNT. Littermates (white columns, n = 3), Nav1.3KO (orange columns, n = 3), Nav1.8KO (light blue columns, n = 3), and Nav1.9KO (turquoise columns, n = 3). (F) Behavioral von Frey responses following SNT surgery on 6-OHDA sympathectomized Nav1.7^Advill (red squares, n = 8) and littermate (purple squares, n = 7) mice, in comparison to unsympathectomized littermate controls (white squares, n = 7). (G) Intraplantar norepinephrine (200 ng) injection sensitizes Nav1.7^Wnt1 mice (black line/green square, n = 7) 14 days after SNT surgery, in comparison to vehicle alone in Nav1.7^Wnt1 mice 14 days after SNT surgery (green line and squares, n = 6). Data analyzed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean ± SEM. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (individual points). See also Figure S2.

Figure 3

Behavioral Responses of Nav1.3, Nav1.8, and Nav1.9 Knockout Mice Reveal Critical Roles in Modality-Specific Responses to CCI-Induced Pain but Not Sympathetically Mediated SNT Pain Behavioral von Frey and acetone responses of Nav1.3, Nav1.8, or Nav1.9 knockouts following CCI (A) and L5 SNT surgery (B). Nav1.3KO mice (orange squares, n = 6) show reduced CCI-induced cold allodynia (Aa) and mechanical allodynia (Ab) in comparison to littermate mice (white squares, n = 10). Nav1.8KO mice (light blue squares, n = 8) show diminished CCI-induced cold allodynia (Ac) but do develop mechanical allodynia (Ad) in comparison to littermate mice (white squares, n = 10). Nav1.9KO mice (turquoise squares, n = 8) show diminished CCI-induced cold allodynia (Ae) but do develop mechanical allodynia (Af) in comparison to littermate mice (white lines, n = 8). Nav1.3KO mice (orange squares, n = 10) develop both L5 SNT-induced cold (Ba) and mechanical allodynia (Bb) in comparison to littermate mice (white squares, n = 8). Nav1.8KO mice (light blue squares, n = 8) develop L5 SNT-induced cold (Bc) and mechanical allodynia (Bd) in comparison to littermate mice (white squares, n = 8). Nav1.9KO mice (turquoise squares, n = 10) develop L5 SNT-induced cold (Be) and mechanical allodynia (Bf) in comparison to littermate mice (white squares, n = 7). Data analyzed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean ± SEM. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (individual points).

Figure 4

Oxaliplatin-Induced Pain and Cancer-Induced Bone Pain Do Not Require Nav1.7 Expression or Nav1.8⁺ Nociceptors Nav1.7^Wnt1 (green squares, n = 7) and littermate (white squares, n = 13) mice treated twice weekly (red arrows) with 3.5 mg/kg oxaliplatin (i.v.) develop both mechanical (A) and cold (B) allodynia. Nav1.8^DTA (black squares, n = 10) and littermate mice (white squares, n = 11) treated twice weekly with 3.5 mg/kg oxaliplatin (i.v.) develop both mechanical (C) and cold (D) allodynia. Limb use scores for the affected hind limb (E) and percentage of body weight placed on the affected hind limb (F) of Nav1.7^Wnt1 (green squares, n = 8) and littermate (white squares, n = 8) mice following cancer induction in the femur. Limb use scores for the affected hind limb (G) and percentage of body weight placed on the affected hind limb (H) of Nav1.8^DTA (black squares, n = 9) and, littermate (white squares, n = 8) mice following cancer induction in the femur. Both Nav1.7^Wnt1 (green column, n = 8) and littermate (white column, n = 8) mice show similar decreases in bone density compared to sham operated mice (black column, n = 5) (I). Example of decreased bone density in a Nav1.7^Wnt1 (J) and littermate (K) mouse, compared to a sham-operated mouse (L). Scale bars represent 2 mm. Data analyzed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean ± SEM. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (individual points). See also Figure S3.

Figure 5

Reversal of CCI-Mediated Mechanical Allodynia after Tamoxifen-Induced Deletion of Nav1.7 Nav1.7^ADERT2 (red squares, n = 8) mice develop mechanical allodynia normally in comparison to littermate controls (white squares, n = 9) following CCI surgery. However, activation of Advillin-CreERT2 through five daily intraperitoneal tamoxifen injections (2 mg per day) reverses this mechanical allodynia in Nav1.7^ADERT2 mice but not Advillin-CreERT2 negative littermate controls. Data analyzed by two-way analysis of variance followed by the Bonferroni post hoc test. Results are presented as mean ± SEM. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 (individual points).