Botulinum toxin B in the sensory afferent: transmitter release, spinal activation, and pain behavior

Abstract

We addressed the hypothesis that intraplantar botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and, over time, will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release, and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1 U) given unilaterally into the hind paw had no effect upon survival or motor function, but ipsilaterally decreased: (1) intraplantar formalin-evoked flinching; (2) intraplantar capsaicin-evoked plasma extravasation in the hind paw measured by Evans blue in the paw; (3) intraplantar formalin-evoked dorsal horn substance P (SP) release (neurokinin 1 [NK1] receptor internalization); (4) intraplantar formalin-evoked dorsal horn neuronal activation (c-fos); (5) ipsilateral dorsal root ganglion (DRG) vesicle-associated membrane protein (VAMP); (6) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; (7) ipsilateral activation of c-fos otherwise evoked bilaterally by intrathecal SP. These results indicate that BoNT-B, after unilateral intraplantar delivery, is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP, and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

Keywords: Botulinum toxin; Dorsal root ganglion; Neurotransmitter release; Sensory afferent; Spinal cord; Substance P; VAMP.

Figure 1

Intraplantar (IPLT) botulinum toxin B (BoNT-B) inhibits formalin-evoked flinching behavior. Plots indicate mean ± SEM for cumulative flinches observed during Phase 1 (0-10min, top) and Phase 2 (11-60min, bottom) following unilateral formalin injection into the hindpaw. As indicated, unilateral IPLT formalin evoked a biphasic flinching response.1A) Mice were pretreated for 24hrs with 0.1, 0.3, 0.5, 1 or 1.5U of IPLT BoNT-B or saline before formalin. IPLT BoNT-B at 1 and 1.5U produced a significant reduction in phase 2 flinching. 1B) Mice received IPLT saline or IPLT BoNT-B (1U) 4 hr, 1d, 7d, or 21 d before formalin (saline data are pooled). Pretreatment with IPLT BoNT-B (1U) for 1d and 7d (but not 4h or 21 days) resulted in a significant reduction in phase 2 flinching. No differences in phase 1 flinching were detected between groups in 1A or 1B. Saline and 1U-1d data are the same in the two graphs. **p<0.01 and ***P<0.001 vs. saline; one-way ANOVA. N=7-8 animals per group.

Figure 2

IPLT-BoNT-B inhibits capsaicin-induced plasma extravasasion in the injected paw. Mice were pretreated with IPLT BoNT-B (1U) and saline (contralateral paw) for 4hr, 1, 7 or 21d and plasma extravasation was measured (Evans blue in the paw tissue) 10 min after bilateral IPLT injection of capsaicin. A) Paw skin color on the saline side (right) is darker than the BoNT-B side (left). B) Plot (mean ± SEM) depicts dye concentration in formamide extract of ipsi- and contralateral paw tissue. C) Plot depicts differences in dye concentration between the contra- and ipsilateral paw. A significant difference was detected between the saline group and the 4hr, 1 and 7d but not the 21d groups. *p<0.05; **p<0.01; ***p<0.001 vs. saline; one-way ANOVA. N=4-6 animals per group.

Figure 3

Intraplantar (IPLT) botulinum toxin B (BoNT-B) inhibits IPLT formalin-evoked NK1 receptor (NK1r) internalization in the spinal dorsal horn. A and B) Representative immunohistochemistry images showing A) a neuron with uninternalized NK1r and B) a neuron with NK1r internalization in the ipsilalateral dorsal horn after IPLT formalin in a control animal. C) Plots indicates percentage of NK1r internalization in the ipsilateral and contralateral superficial layer of the lumbar spinal dorsal horn. IPLT formalin evokes an increase in the incidence of NK1r Internalization in the ipsilateral, but not contralateral dorsal horn in the L4, L5 and L6 spinal segments. Pretreatment with IPLT BoNT-B (1U) for 4 hr, 1 and 7d but not 21d prevented Nk1r internalization. *p<0.05; ** p<0.01 vs. saline; one-way ANOVA. N=3-6 animals per group with 9-12 spinal cord sections counted per animal. Scale bar, 15μm.

Figure 4

Intraplantar (IPLT) botulinum toxin B (BoNT-B) inhibits IPLT formalin-induced cFos expression in the spinal dorsal horn. The plot presents mean ± SEM of number of cFos (+) neurons in the spinal dorsal horn in the L4, L5 and L6 segments. IPLT formalin evokes a unilateral increase in cFos(+) neurons in the ipsi- but not contralateral dorsal horn. This formalin-induced cFos increase was blocked by pretreatment with IPLT BoNT-B (1U) for 1 and 7d but not 4hr and 21d. ** p<0.01 vs. saline; one-way ANOVA. N=3 animals per group with 9-12 spinal cord sections counted per animal.

Figure 5

Intraplantar (IPLT) botulinum toxin B (BoNT-B) reduces vesicle associated membrane protein (VAMP) expression in the ipsilateral DRG. A) Western blots showing VAMP and -actin (loading control) in the DRG and spinal cord ipsi- and contralateral to IPLT injection of BoNT-B (1U). Mice were treated with IPLT BoNT-B for 4h, 1, 7 or 21 days prior to tissue harvest. B) Histogram depicts the relative levels of VAMP expression quantified by densitometric measurement. Saline control tissues (set as 100%, not shown) were used for normalization. A significant decrease in VAMP expression in the ipsilateral DRG was observed after 1d and 7d BoNT-B pretreatment. Values are mean ± SEM. *P < 0.05; **P < 0.01 vs. the contralateral side; one-way ANOVA. N=3-4 animals per group.

Figure 6

Intraplantar (IPLT) botulinum toxin B (BoNT-B) inhibits intrathecal capsaicin-evoked NK1 receptor (NK1r) internalization in the spinal dorsal horn. Plot presents the percentage of NK1r(+) neurons displaying NK1r internalization in the superficial dorsal horn after intrathecal delivery of capsaicin (10μg/10μL). Mice were pretreated with unilateral IPLT saline or BoNT-B (1U/30μL) 1 day in advance of the intrathecal capsaicin. Both groups received IT capsaicin. In saline control mice, IT capsaicin induced bi-lateral NK1r internalization. IPLT BoNT-B pretreatment blocks NK1r internalization on the ipsi- but not contralateral side. p<0.01, ipsi vs. contra-lateral; paired t-test.

Figure 7

Representative Immunolabeling of cFos and NK1r in the lumbar L4 spinal cord dorsal horn of animals which received unilateral hindpaw intraplantar (IPLT) BoNT-B or saline control, then IT substance P after 48 hrs. A) In control animals received IPLT saline, IT SP induced a bilateral increase of cFos(+) profiles in the dorsal horn. B) IT SP resulted in an increased incidence of cFos(+) neurons in the dorsal horn contralateral but not ipsilateral to the IPLT BoNT. C) Scatter plot shows the numerical differences of cFos profiles between the contralateral and ipsilateral dorsal horn. There was a significant difference between the BoNT-B group and the control group. ****P<0.0001 vs. the contralateral side; one-way ANOVA. D, E and F) Double labeling of D) c-fos and E) NK1r in the spinal cord of a mouse received IPLT BoNT-B and IT SP. G-R) Higher magnification images of the two areas in contralateral (con1 and con2) and ipsilateral (ipsi1 and ipsi2) dorsal horns shown in panel F) outlined by the boxes. In con1 and con2, a combined total of 26 c-fos profiles were counted. * denotes 5 neurons (19%) that are c-fos (+) and NK1r (+). On the ipsilateral side, there was a much lower number of c-fos(+) profiles and no c-fos(+)/NK1r(+) profiles were noted. D-R) Images from a single focal plane were shown. Scale bars, A, B, D-F, 250μm; G-R, 50μm.