Intra-paw injections of FLO inhibited heat nociception in naive wild-type (WT) mice and attenuated both heat and mechanical hyperalgesia after the plantar-incision.

(A) Paw withdrawal latency (PWL) to heat stimulation in naïve WT mice before and after injection of FLO (0.5 mg, 20 μL) or the vehicle (saline, 20 μL) into the dorsum of the hind paw. Ipsilateral: injected side; Contralateral: un-injected side. N=10/group. (B) The PWL ipsilateral to the side of plantar-incision was measured before and 1, 2, 4 hours after intra-paw injections of FLO (0.1 mg, 0.5 mg, 20 μL) or the vehicle in WT mice during Days 2-4 post-injury. N=7-13/group. (C) The mechanical paw withdrawal threshold (PWT) to noxious pinch applied to the side of plantar-incision was measured before and 1 hour after intra-paw injection of FLO (0.5 mg, 20 μL) or vehicle with the Randall-Selitto test during Days 2-4 post-injury. N=10-11/group. (D) Schematic of the Catwalk gait analysis (left) and the representative paw print images (right). (E) Quantification of print area and maximum contact area in Catwalk test before and 1 hour after an intra-paw injection of FLO (0.5 mg, 20 μL) or vehicle on Day 2 post-injury. The left hind paw (LH) received the incision and drug treatment, and data were normalized to the right side (RH). N=9-10/group. (F) Locomotor function and exploration were assessed in the open field test (30-min duration). The number of total, central and peripheral beam breaks were measured before and at 1 hour after an intra-paw injection of FLO (0.5 mg, 20 μL) or vehicle during Days 2-4 post-injury. N=10/group. Data are mean ± SEM. Two-way mixed model ANOVA followed by Bonferroni post hoc test. (A-C) *P<0.05, **P <0.01, ***P <0.001 versus vehicle; #P<0.05, ##P<0.01, ###P<0.001 versus baseline (A) or pre-drug (B, C). (E, F) *P<0.05, **P <0.01 versus pre-drug; ###P<0.001, ####P<0.0001 versus baseline. ns = not significant.

FLO acutely attenuated the responses of small DRG neurons to noxious heat stimulation.

(A) Upper: Strategy for generating Pirt-Cre; GCaMP6s mice. Lower: Schematic diagram illustrates the experimental setup for in vivo optical imaging of L4 DRG neurons and applying test stimulation. (B-C) Upper: Representative images of calcium transients in DRG neurons in response to noxious heat stimulation (51°C water bath) applied to the hind paw before and 1 hour after an intra-paw injection of vehicle (B, saline) or FLO (C, 0.5 mg, 20 μL) at Day 2 after plantar-incision. Lower: Percentages of small-, medium- and large-size neurons that were activated (ΔF/F ≥ 30%) by heat stimulation before and after vehicle or FLO. DRG neurons were categorized according to cell body size as <450 μm2 (small), 450–700 μm2 (medium), and >700 μm2 (large). N=9 /group. (D) The higher-magnification representative images (upper) and calcium transient traces (lower) show increased fluorescence intensities in four DRG neurons (indicated by colored arrows) responding to heat stimulation, and decreased responses after FLO treatment. Data are mean ± SEM. (B, C) Paired t-test. ***P<0.001 versus pre-drug.

HC-HA/PTX3 inhibited heat hypersensitivity in wild-type (WT) mice after plantar-incision and attenuated DRG neuron activation.

(A) Left: intra-paw injection of HC-HA/PTX3 (10 μg or 20 μg, 20 μL), but not vehicle (saline), increased paw withdrawal latency (PWL) to heat stimulation in naïve WT mice. N=8-11/group. Right: intra-paw injection of HC-HA/PTX3 (10 μg or 20 μg, 20 μL) dose-dependently attenuated the heat hypersensitivity during Days 2-4 after plantar-incision. N=9-16/group. (B) Right: intra-paw injection of HC-HA/PTX3 (20 μg, 20 μL) showed superior anti-hyperalgesic effect compared to HMW-HA ((20 μg, 20 μL)) alone and the mixture of HMW-HA (20 μg) and HC1 (720 ng) during days 2-4 after plantar-incision. Left: analyzing the Area Under the Curve (AUC) to assess the anti-hyperalgesic effect of each group. N=5-9/group. (C) HC-HA/PTX3 inhibited the calcium responses evoked by capsaicin (a TRPV1 agonist, 0.3 μM) in WT DRG neurons. HC-HA/PTX3 alone did not evoke [Ca2+]i elevation. Pre-treatment (20 min) of HC-HA/PTX3 (15 μg/mL, bath application) reduced capsaicin-evoked [Ca2+]i rising. (D) The quantification of [Ca2+]i rising evoked by capsaicin in DRG neurons pre-treated with the vehicle, HC-HA/PTX3 (15 μg/mL), or HMW-HA (15 μg/mL). N=109-170 neurons/group. (E) Left: Traces show that the β-alanine (a MrgprD agonist, 1 mM) evoked an increase in [Ca2+]i, which was also inhibited by HC-HA/PTX3. Right: The quantification of evoke [Ca2+]i rising by β-alanine. N=10-25 neurons/group. (F) Left: Traces show that cinnamaldehyde (a TRPA1 agonist, 1 mM) evoked an increase in [Ca2+]i, which was inhibited by HC-HA/PTX3. Right: The quantification of evoke [Ca2+]i rising by cinnamaldehyde. N=15-35 neurons/group. (G) An example trace of membrane potential (Vm) which changed from resting level (−60 mV) toward a more hyperpolarized state after HC-HA/PTX3 (10 μg/mL) in a small DRG neuron (insert, scale bar: 25 μm). Vm returned to pre-drug level after washout. DRG neurons were categorized according to cell body diameter as <20 μm (small), 20–30 μm (medium), and >30 μm (large). (H) Example traces of action potentials (APs) evoked by injection of current in small DRG neurons 5 min after bath application of vehicle or HC-HA/PTX3 (5, 25 μg/mL). (I) HC-HA/PTX3 concentration-dependently altered the intrinsic membrane properties of small DRG neurons. Quantification of the resting membrane potential (RMP) before and at 5 min after bath application of vehicle or HC-HA/PTX3 (5, 10, 25 μg/mL). N=4-7/group. (J) Quantification of rheobase in small DRG neurons at 5 min after vehicle or HC-HA/PTX3. The rheobase after drug was normalized to pre-drug value. N=5-7/group. Data are mean ± SEM. (A, B: right) Two-way mixed model ANOVA followed by Bonferroni post hoc test. *P< 0.05, **P<0.01, ***P<0.001 versus vehicle; #P<0.05, ##P<0.01, ###P<0.001 versus pre-drug. (B: left, C) One-way ANOVA followed by Bonferroni post hoc test. ***P<0.001 versus vehicle; ##P<0.01 versus other groups. (E, F) Paired t-test. ***P<0.001 versus vehicle. (I, J) Two-way mixed model ANOVA followed by Bonferroni post hoc test. *P<0.05, **P<0.01 versus pre-drug.

FLO and HC-HA/PTX3 inhibited pain via CD44-dependent mechanisms.

(A) The expression of CD44 in the DRG of wild-type (WT) mice. Left: Colocalization of CD44 and CGRP (a), IB4 (b) and NF200 (c) immunoreactivity (IR). Right: The quantification of CD44-expressing neurons (as % of total neurons in each subpopulation, IB4+: 96%; CGRP+: 82%; NF200+: 68%, N=4) (d) The size distribution of CD44-expressing neurons. (B) Left: Dot plot of CD44 gene expression in different clusters [SGC (1), NF (2), NP (3), PEP (6), cLTMR (1)] of DRG cells from WT mice in single-cell RNA-sequencing study. The dot size represents the percentage of cells expressing CD44, and the color scale indicates the average normalized expression level. The NF1 and NF2 clusters were indicated with a red circle. Right: Violin plot shows the CD44 expression levels in each cluster. SGC: satellite glial cells; NF, Aβ or Aδ low-threshold mechanoreceptors or proprioceptors; NP, non-peptidergic nociceptors or pruriceptors; PEP, peptidergic nociceptors; C-LTMR, C-fiber low-threshold mechanoreceptors. One-way ANOVA followed by Bonferroni post hoc test. **P<0.01 ***P<0.001 versus NF1; #P<0.05, ##P<0.01, ###P<0.001 versus NF2. (C) Traces show that the capsaicin (0.3 μM) evoked an increase of [Ca2+]i in a small neuron from CD44 KO mouse. Compared to [Ca2+]i rising evoked by the 1st capsaicin application, there was a reduction of [Ca2+]i rising to the 2nd treatment, indicating TRPV1 desensitization. DRG neurons were categorized according to cell body diameter as <20 μm (small), 20–30 μm (medium), and >30 μm (large). (D) Capsaicin-evoked increases of [Ca2+]i before and after treatment (20 min) with HC-HA/PTX3 (10 μg/mL) in small DRG neurons from CD44 KO mice.(E) The quantification of evoked [Ca2+]i rising by capsaicin. HC-HA/PTX3 pretreatment did not reduce capsaicin-evoked [Ca2+]i rising in CD44 KO neurons. N=100-120 neurons/group. (F) HC-HA/PTX3 did not change the intrinsic membrane property of small DRG neurons from CD44 KO mice. Upper: An example trace of membrane potential (Vm) which remained around resting level (−60 mV) after HC-HA/PTX3 (10 μg/mL). Lower: Quantification of the resting membrane potential (RMP) at 5 min after vehicle (saline) and HC-HA/PTX3 (P=0.48). (G) Upper: Examples traces of action potentials and rheobase evoked by injection of current in a small CD44 KO DRG neuron at 5 min after vehicle or HC-HA/PTX3 (10 μg/mL). Lower: Quantification of the rheobase levels (P=0.2), action potential (AP) threshold (P = 0.87), AP amplitude (P=0.75) and duration (P=0.82) in small DRG neurons from CD44 KO mice. N=7-11/group. (H) Paw withdrawal latency (PWL) that was ipsilateral to the side of plantar-incision before and after an intra-paw injection of FLO (0.5 mg, 20 μL) or vehicle (saline, 20 μL) in CD44 KO mice (H, N=8-9/group) after plantar-incision. (I) The ipsilateral PWL before and after an intra-paw injection of HC-HA/PTX3 (10 μg or 20 μg, 20 μL) or vehicle in CD44 KO mice after plantar-incision. N=7-9/group. (J) The ipsilateral PWL before and after intra-paw injection of vehicle + control IgG, vehicle + CD44 IgG, HC-HA/PTX3 (10 μg) + control IgG or HC-HA/PTX3 (10 μg) + CD44 IgG (all IgG at 10 μg, 10 μL) in WT mice after plantar-incision. N=8-11/ group. Data are mean ± SEM. (E) One-way ANOVA followed by Bonferroni post hoc test. *P<0.05 versus WT vehicle. ns=not significant. (F, G) Student’s t-test. (H-K) Two-way mixed model ANOVA followed by Bonferroni post hoc test. **P<0.01 versus vehicle or saline + IgG; #P<0.05 versus pre-drug.

HC-HA/PTX3 induced cytoskeletal rearrangement which contributes to its inhibition of INav and HVA ICa.

(A) Example images show the distribution of F-actin and CD44 staining in small DRG neurons of wild-type (WT) mice. Neurons were treated with bath application of vehicle (saline), HMW-HA (15 μg/mL), HC-HA/PTX3 (10, 15 μg/mL), or HC-HA/PTX3 (10, 15 μg/mL) combined with Latrunculin A (LAT-A, 1 μM) for 45 min. Scale bar: 5 μm. DRG neurons were categorized as <20 μm (small), 20–30 μm (medium), and >30 μm (large). (B) Quantification of submembranous F-actin polymerization and translocation of CD44 in small WT DRG neurons after drug treatment. N=30-80/group. (C) Proliferation MTT assay showed a lack of neuronal toxicity from 0.5, 1, 2, 5, 10, 15 μg/mL HC-HA/PTX3, compared to vehicle (100% viable cells). N=6-12 repetitions/group.(D) Quantification of submembranous F-actin polymerization and translocation of CD44 in small DRG neurons. DRG neurons were electroporated with siRNA targeting Pfn1 (siPfn1) or non-targeting siRNA (siNT, control). Neurons were treated with vehicle (saline) or HC-HA/PTX3 (10 μg/mL) for 45 min. N=70-111/group. (E) Changes in the submembrane distribution of F-actin and CD44 in WT DRG neurons treated with vehicle + control IgG (2 µg/mL), HC-HA/PTX3 (15 μg/mL) + control IgG (2 µg/mL), or HC-HA/PTX3 (15 μg/mL) + CD44 IgG (2 µg/mL) for 45 min. Scale bar: 5 μm.(F) Quantification of the submembrane F-actin and CD44 labeling in each group. (G) Infusion of LAT-A attenuated the inhibition of INav by HC-HA/PTX3 in WT DRG neurons. a. Representative traces of INav after 5 min infusions of vehicle (top row) or LAT-A (bottom row, 0.5 nM) through the recording electrode, followed by bath application of HC-HA/PTX3 (10 µg/mL). Lumbar DRG neurons were harvested on day 2-3 after plantar-incision. b. There was a significant interaction between the variation produced by HC-HA/PTX3 (10 µg/mL) and test voltages (VTest) applied in vehicle-infused neurons, resulting in an overall INav inhibition (F(14,90) = 3.29, ***P<0.001), and significantly decreased INav density (pA/pF) from VTest = −10 mV to +10 mV, as compared to pre-HC-HA/PTX3 treatment. N=7/group. c. HC-HA/PTX3 did not alter GNa/GNa max across the test voltages (F(9,60) = 0.44, P=0.9) in vehicle-infused neurons. N=7/group. d. There was a significant interaction between the variation produced by HC-HA/PTX3 (10 µg/mL) and VTest applied in LAT-A-infused neurons, resulting in overall INav increase (F(14,120) = 1.87, *P<0.05) and increased INav density (pA/pF) from VTest = −10 mV to 0 mV, as compared to pre-HC-HA/PTX3. N=9/group. e. HC-HA/PTX3 significantly increased the GNa/GNa max at VTest= −20 mV in LAT-A-infused neurons (*P<0.05, N=9/group).(H) LAT-A attenuated the inhibition of HVA-ICa by HC-HA/PTX3 in WT DRG neurons. a. Representative traces of HVA-ICa in small WT DRG neurons after 5 min infusions of vehicle (top row) or LAT-A (bottom row, 0.5 nM), followed by bath application of HC-HA/PTX3 (10 µg/mL). b. In vehicle-infused neurons, HC-HA/PTX3 (10 µg/mL) significantly decreased HVA-ICa (F(1,12)=6.52, *P=0.02) and HVA-ICa conductance (I/Imax) from VTest = −40 mV to +10 mV, as compared to pre-HC-HA/PTX3. N = 7. c. HC-HA/PTX3 did not alter the channel open probability (Po) in vehicle-infused neurons (P=0.82, N=7). d. In LAT-A-infused neurons, HC-HA/PTX3 only modestly reduced HVA-ICa conductance across test voltages applied (F(1,12)=0.27, P=0.6, N=8). e. HC-HA/PTX3 did not alter Po in LAT-A-infused neurons (P=0.94, N=8). Data are mean ± SEM. (B, C, D, F) One-way ANOVA followed by Bonferroni post hoc test. *P< 0.05, ***P<0.001 versus vehicle; #P<0.05, ###P<0.001 versus indicated group. (G, H) Two-way repeated measures ANOVA with Holm-Sidak post-test. *P< 0.05, **P< 0.01, ***P<0.001, ****P<0.0001 versus vehicle infusion or LAT-A infusion group.