Abstract
Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.
SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.
Introduction
Despite many available analgesics, incisional pain remains a significant challenge in the perioperative period. Opioids are commonly used to control acute postoperative pain, but there are many side effects which can limit their use, including nausea, vomiting, and constipation. Moreover, prescription opioids which are commonly given for acute postoperative pain on discharge have an increased risk of persistent opioid use and contributory to the current opioid epidemic (Carroll et al., 2012; Clarke, 2016; Soneji and Peng, 2016).
One potential alternative approach to opioids is the prophylactic use of capsaicin, a compound extracted from chili peppers (genus Capsicum). Upon initial application, capsaicin evokes pain and hyperalgesia because of the opening of non-selective cation channels located on small diameter neurons expressing TRPV1 receptors. Capsaicin reduced visual analog scores (VAS) in humans and pain-related behaviors in animal models of incisional pain (Hamalainen et al., 2009; Hartrick et al., 2011; Uhelski et al., 2020).The intraplantar capsaicin pretreatment before incision alleviates spontaneous and thermal pain because of degeneration of CGRP and IB4/PGP9.5 positive nociceptive nerve fibers in the skin of rats (Kang et al., 2010). The genome-wide transcriptional profiling of dorsal root ganglia (DRG) tissues in a rat model of incisional pain, identified changes in gene expression in the DRG at 1 d after plantar incision, a time point at which pain behaviors were most prominent. The skin infiltration of capsaicin 30 min before surgical incision attenuated incision-induced pain behaviors, completely denervated nerve fibers at the epidermis and dermis around the incision, and prevented incision-induced transcriptional changes in 99 of 126 DRG genes (79%; Tran et al., 2020).
Despite these attractive findings and studies, in vivo DRG activity at the population level is unknown, and further study might give us a clue to better determine candidate molecules for targeting incisional pain. We therefore developed in vivo primary sensory neuronal cell body Ca2+ imaging using our Pirt-GCaMP3 animal model, in which Ca2+ activities of >1800 neurons in the DRG can be visualized (Kim et al., 2016). Furthermore, Pirt-GCaMP3 enables us to monitor primary sensory nerve fibers in the isolated skin despite being surrounded by many other cell types (Kim et al., 2014). This model permits us to monitor and study Ca2+ movement in almost all primary sensory neuron cell bodies and fibers using our Pirt-GCaMP3 animals. In the present study, we have assessed in vivo primary sensory neuronal cell body Ca2+ activity in DRG on a populational ensemble level, and in situ Ca2+ activity in primary sensory nerve fibers in sliced spinal cord in the incisional pain model.
Materials and Methods
Animals
Mice (C57BL/6J, The Jackson Laboratory) were housed in a regular light-dark cycle, with food and water available ad libitum. The animal use protocol was approved by the Institutional Animal Care and Use Committee and according to the guidelines for animal experiments approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio (UTHSA). Male and female mice were used for all experiments.
Postoperative (incisional) pain model
One week after intraplantar injection of capsaicin or vehicle, a 5-mm longitudinal incision was made with a number 11 blade through the skin, fascia, and muscle of right hindpaw under conditions of anesthesia with 2–3% isoflurane. The incision was closed with two single sutures of 6–0 nylon and triple antibiotic ointment was applied.
Drug treatment
Capsaicin (Sigma) was dissolved in vehicle (1.5% Tween 80, 1.5% ethanol, and 97% saline). Fifty microliters of 0.05% capsaicin (100 μg/200 μl) or an equal volume of vehicle was subcutaneously injected to the incision site of the right hindpaw using a 0.5-ml insulin syringe with a 28-gauge needle under conditions of anesthetization using ketamine hydrochloride (80 mg/ml)/xylazine hydrochloride (12 mg/ml) solution, 10 µl/10 g weight. Capsaicin was subcutaneously injected into the intraplantar area 7 d before incision surgery.
Measurement of 50% paw withdrawal threshold
von Frey filament was applied to plantar surface and escape behavior was observed. A mouse was placed in a transparent plastic cage on a metal grid and allowed to habituate for at least 1 h before testing. von Frey filament intensity from 0.04 g (no. 2.44) to 2.0 g (no. 4.31) was used with an up-down test. The filament was bent for 3 s after contacting to plantar skin. The interval between each application was 3 min after escape behavior was observed, or 30 s if escape behavior was not observed. To calculate the 50% threshold of paw withdrawal, the following formula was used: 10(xf+kx0.22)/10,000. Xf = the value of the final filament used (in log units), k = a value based on the response pattern, as reported by Chaplan et al. (1994).
Measurement of thermal pain test
To measure the thermal response, a Hargreaves test was performed. A mouse in a transparent plastic cage on heated glass board at 30°C was allowed to habituate for at least 1 h before testing. Radiant heat was applied to the mouse plantar area through the glass, and the duration until escape behavior was measured. Initial heat applied was 34°C, which increased to 51°C in 10 s. This process was done five times, and the average time to escape behavior was calculated. The interval between each trial was at least 5 min.
Measurement of spontaneous foot lifting
Spontaneous foot lifting was used as a measure of spontaneous pain. A mouse in a transparent plastic cage on a metal grid was allowed to habituate for at least 1 h before testing. An angled mirror was set under the metal grid during habituation and mouse behavior reflected by the mirror was recorded with video camera for 10 min. Frequency of spontaneous foot lifting and foot licking of the incision foot was determined.
Open field test
Three hours after incision surgery, an open field test was performed. To monitor mouse behavior, a mouse was put in a new cage surrounded by white square box. The distance between the white board and side of the cage was around 10 cm. Behaviors were recorded for 10 min using a camera mounted above the cage, and were assessed as total walking distance. Movement was calculated using ImageJ software with the Animal Tracker plugin (Gulyás et al., 2016).
In vivo DRG Ca2+ imaging
One day after incision surgery, in vivo Ca2+ imaging in the DRG was performed. L5 entire DRG Ca2+ imaging in a live mouse was performed for 2–4 h after DRG exposure as we previously described (Kim et al., 2016). Rectal temperature was monitored as body temperature and kept at around 37°C using a heat pad. Mouse movement because of breath and heart beat was minimized using clamps on vertebra bone. The mouse was anesthetized with ketamine hydrochloride (80 mg/ml)/xylazine hydrochloride (12 mg/ml) solution mixed with saline (1:1), 10 µl/10 g weight. Live images were obtained using single photon confocal microscopy (Carl Zeiss) from 10 frames at 465–960 ms/frame in each z-axis (512 × 512 or 1024 × 1024 pixels in the x-y plane) using a 10× dry objective lens. Solid diode lasers at 488- and 532-nm wavelength were used for emission at 500–550 nm for green fluorescence and 550–650 nm for red fluorescence, respectively. The depth of each x-y focal plane where the laser captured images was from 0 to ∼100 µm. Fifteen image cycles were obtained, and the duration time of each cycle was around 4.5–7.5 s. Small brush, large brush, 0.4 g, and 2.0 g von Frey filament were applied to the hindpaw of exposed DRG side. A press of 100 and 300 g were applied to the whole palm of hindpaw using a rodent pincher (Bioseb). Whole hindpaw was immersed into 50°C water, and acetone was applied by pipette to the hindpaw. One hundred mM KCl (30 μl) was subcutaneously injected into the hindpaw using a 0.5-ml insulin syringe with a 28-gauge needle. Small and large brush bristles were 5 and 40 mm, respectively.
In situ Ca2+ imaging in spinal cord slice
Animals were killed by decapitation under conditions of anesthesia using ketamine. The lumbar enlargement area (L3–5) was removed, and placed in ice-cold NMDG solution (135 mm N-methyl-D-glucamine, 1 mm KCl, 1.5 mm MgCl2, 1.2 mm KH2PO4, 0.5 CaCl2·2H2O, 29.5 mm C5H14NO·HCO3, and 10.7 mm glucose) aerated with (5% CO2, 95%O2). Spinal cord was dissected and transversely sliced at 300–400 µm using a Leica VT1200S vibratome at 4°C. The spinal cord slices were immediately placed in synthetic interstitial fluid (107.8 mm NaCl2, 3.5 mm KCl, 0.69 mm MgSO4·7H2O, 1.67 mm NaH2PO4·2H2O, 5.55 mm glucose, 7.6 mm sucrose, 1.53 mm CaCl2·2H2O, 26.2 mm NaHCO3, and 9.64 mm gluconic acid sodium salt) aerated with (5% CO2, 95%O2) on an imaging stage (Siskiyou Corporation) stabilized with Harp (Scientific Instruments). The dorsal horn of the tissue was viewed using a 40× water immersion objective lens (Carl Zeiss). The z-axis of spinal cord was imaged at a depth of 100–150 μm at ∼10-µm intervals. A solid diode laser at 488 nm was used for emission at 500–550 nm for green fluorescence. During Ca2+ imaging experiments at room temperature, synthetic interstitial fluid solution aerated with 95% O2/5% CO2 was perfused at 400 ml/h. 2-D images of primary sensory nerve fiber were made from merged z-axis maximum fluorescence intensities (512 × 512 pixels; Kim et al., 2014). Green fluorescence from GCaMP3 was analyzed by ImageJ (NIH).
Ca2+ transient calculation
Ca2+ transients in in vivo DRG and in spinal cord slice imaging were determined using the formula: ΔF/F0, where F0 represents basal intensity at 500–550 nm for green fluorescence, and ΔF represents intensity calculated by subtracting basal intensity from the intensity at each time point.
Cell size measurement
Cell size was defined by the average of the long diameter and short diameter from one cell body using Zen Blue software (ZEN3.1).
Immunohistochemistry
Plantar skin was dissected 1 d after surgery, and washed in 0.1 m PBS followed by 4% paraformaldehyde (PFA) overnight at 4°C. Tissue was transferred to 10% sucrose overnight at 4°C followed by 30% sucrose, and frozen at −80°C. Frozen skin was returned to room temperature, transferred to optimal cutting temperature (OCT) embedding medium (Fisher Healthcare), and frozen at −20°C. Tissue was sliced (20 µm) using a cryostat. Sections were transferred to slides and dried at 37°C for 1 h, incubated with guinea pig anti-TRPV1 antibody (1:250, catalogue number ab10295, Abcam), chicken anti-GFP antibody (1:250, catalogue number CH23105, Neuromics), and rabbit anti-β-III tubulin antibody (1:250, catalogue number ab18207, Abcam) overnight at 4°C incubation of blocking solution. Primary antibody was washed out with PBS, and slides were incubated for 90 min at room temperature with goat secondary antibody to guinea pig IgG-Alexa Fluor 568 (1:200, Invitrogen), goat secondary antibody to Rabbit IgG-H&L (Cy5; 1:200, catalog #A-21244) and goat secondary antibody to chicken IgG Alexa Fluor 488 (1:200, Invitrogen). Sections were washed three times in PBS, and a cover glass was applied with Prolong Diamond Antifade Mountant with DAPI (Invitrogen). Immunofluorescence was viewed using a 10× dry or 40× water immersion objective lens by single photon confocal microscopy (Carl Zeiss). Human placental alkaline phosphatase (PLAP) is a useful protein to trace peripheral axons in dissected tissue and whole mount skin. We purchased PLAP-floxed mice from The Jackson Laboratory, and used the experimental method described by Jeremy Nathans at Johns Hopkins University (Chang et al., 2014). After 4% PFA fixation, skin tissue was fixed on sylgard in a 10-cm glass dish, and then transferred into PBS containing 1 mm MgCl2. The dish was gently rotated in a water bath at 70°C for 90 min. Skin tissue was transferred to BCIP/NBT membrane alkaline phosphatase substrate solution (Rockland), and incubated around 36 h at room temperature. To stop the reaction, skin tissue was washed tree times with PBS/0.1% Tween 20 over 1 h. After washing, skin tissue was moved to ethanol to dehydrate, and then pinned on sylgard with benzyl benzoate and benzyl alcohol (BBBA) for 3 h. Skin tissue was placed between glass plates, and any bubbles and extra BBBA were removed. PLAP staining was imaged using a bright-field microscope with 10× and 40× objectives.
Drugs
Capsaicin was purchased from Sigma-Aldrich, and wortmannin was purchased from AdooQ science; other drugs were purchased from Fisher Bioreagents.
Statistical analysis
Results are expressed as mean ± SEM. Student's t test was used for single comparisons. For multiple comparisons, two-way ANOVA followed by Student's t test with Bonferroni correction or one-way ANOVA followed by Dunnett's test were used to analyze differences. If no difference was detected by ANOVA, no multiple comparisons were performed; p < 0.05 was considered statistically significant. All statistical analysis was performed on GraphPad Prism 9.
Results
Administration of capsaicin before surgical incision attenuated spontaneous pain behavior and prevented heat and mechanical hyperalgesia
An incision of the plantar area induced mechanical and thermal pain that was assessed at 3 h, 1 d, 2 d, and 3 d after the surgery (Fig. 1A: F(1,66) = 94.46, p = 0.0001, ANOVA; Fig. 1B: F(1,54) = 48.98, p = 0.0001, ANOVA; Fig. 1C: F(1,66) = 65.87, p = 0.001, ANOVA; Fig. 1D: F(1,66) = 121.9, p = 0.001, ANOVA). The injection of capsaicin 7 d before the incision surgery alleviated the mechanical and thermal pain induced by the incision surgery (Fig. 1E: F(1,77) = 20.20, p = 0.0001, ANOVA; Fig. 1F: F(1,77) = 53.07, p = 0.0001, ANOVA). The injection of capsaicin also decreased spontaneous foot lifting following incision surgery (F(1,25) = 17.25, p = 0.0003, ANOVA; Fig. 2A). In open field tests, total walking distance and average walking velocity from incised mice were reduced compared with naive and capsaicin-treated mice following the incision (Cap-ICS mice; Fig. 2C: q(15) = 3.922, p = 0.0026, Dunnett's test incision vs naive; q(15) = 5.689, p = 0.0001, Dunnett's test incision vs ics-capsaicin; Fig. 2D: q(15) = 3.945, p = 0.0024, Dunnett's test incision vs naive; q(15) = 4.121, p = 0.0017, Dunnett's test incision vs ics-capsaicin).
Capsaicin pretreatment (intraplantar injection, 0.05% capsaicin) significantly alleviates postoperative mechanical and thermal pain in a mouse model. A, B, Mechanical pain test using von Frey filament was performed after incision surgery at 3 h, 1 d, 2 d, 3 d, and 7 d in male and female mice. Mechanical sensitivity is plotted as 50% withdraw threshold in grams. C, D, Thermal pain test using Hargreaves method was performed after incision surgery at 3 h, 1 d, 2 d, 3 d, and 7 d in male and female mice. Thermal sensitivity is plotted as paw withdraw latency following thermal stimulus. E, F, Mechanical or thermal pain test using von Frey filament or Hargreaves method was performed after incision surgery at 3 h, 1 d, 2 d, 3 d, and 7 d in vehicle or capsaicin-treated (7 d before incision surgery) incision mouse model. Cap-ICS: capsaicin-treated incision. A, C, Naive: n = 5, incision: n = 7; B, D, Naive: n = 5, incision: n = 6; E, F, Vehicle: n = 5, Cap-ICS: n = 5. Data represent mean ± SEM; t test, *p < 0.05, **p < 0.01, ***p < 0.001 incision versus naive or vehicle.
Capsaicin pretreatment (intraplantar injection, 0.05% capsaicin) significantly alleviates spontaneous pain and decreases the length of peripheral primary sensory nerve fiber in the skin. A, Spontaneous foot lifting as an index of spontaneous pain was counted at 3 h and 1 d after incision surgery. The number of spontaneous foot lifts by vehicle and capsaicin-treated (7 d before incision surgery) mice is plotted. B, Examples of traces of mouse movement in the open field test for assessment of spontaneous pain in naive, vehicle-treated mice, and capsaicin-treated mice. C, Total walking distance in open field test of naive, vehicle-treated, and capsaicin-treated mice. D, Average walking velocity in the open field test of naive, vehicle-treated, and capsaicin-treated mice. E, Confocal microscopic immunohistochemistry image (40×) of β-III tubulin (sensory nerve fiber), TRPV1 channel, and GCaMP (GFP)-expressing primary sensory neuron fibers in skin from incision and capsaicin-treated Pirt-GCaMP3 mice. Area 1, skin surface; area 2, epidermis; area 3, dermis. Arrowheads indicate nerve fibers and nerve endings in epidermis. F, PLAP staining image (40×) in skin from vehicle-treated and capsaicin-treated Pirt-cre/PLAPflox mice. Black lines are sensory nerve fibers. G, Graph of PLAP-positive skin nerve fiber length and ratio of fluorescence intensities of β-III tubulin and TRPV1 channels from either incision only mice or capsaicin-treated incision mice. Cap-ICS, capsaicin-treated incision. Scale bars: 100 µm. A, Incision, n = 6; Cap-ICS, n = 9. B–D, Naive, n = 5; incision, n = 5; Cap-ICS, n = 5. E, G, Incision, n = 3; Cap-ICS, n = 3. F, G, Incision, n = 3; Cap-ICS, n = 3. Data represent mean ± SEM; t test or Dunnett's test; *p < 0.05, **p < 0.01, ***p < 0.001 incision versus naive or capsaicin-treated incision.
The length and intensity of TRPV1 and PLAP-positive primary sensory nerve fibers are decreased in the capsaicin-treated incision model
We performed intraplantar injection of capsaicin to induce degeneration of TRPV1-positive nociceptive primary sensory nerve fibers in plantar skin. To check the effect of capsaicin injection, expression of transient receptor potential vanilloid (TRPV1) channel-positive primary sensory nerve fibers were measured by IHC. TRPV1-positive primary sensory nerve fibers in the incision skin were decreased by the intraplantar capsaicin injection compared with control incisions (Fig. 2E,G). Furthermore, β-III tubulin-positive skin nerve fibers in capsaicin-treated mice were decreased compared with untreated mice in the incision model (Fig. 2E,G; Fig. 2G: β-III tubulin, t(4) = 10.73, p = 0.0004, t test; TrpV1, t(4) = 5.109, p = 0.0007, t test). Strong PLAP expression in primary sensory nerve fibers are detected in almost all primary sensory nerve fibers in the skin (Wu et al., 2012). We tried to visualize PLAP expression driven by Pirt-cre in all primary sensory nerve fibers using skin flat mount PLAP staining. Primary sensory nerve fiber lengths in capsaicin-treated incision model were dramatically decreased compared with vehicle-treated incision mice (Fig. 2F,G; Fig. 2G: length, t(4) = 3.682, p = 0.0212, t test). Morphologic changes in primary sensory nerve fibers in capsaicin-treated incision mice were detected, showing weak staining and fewer branching fibers than incised controls (Fig. 2F,G).
Plantar incision causes spontaneous Ca2+ activity which is dramatically reduced by injection of capsaicin in vivo in Pirt-GCaMP3 mice
To elucidate the relationship between spontaneous pain and primary sensory neuron Ca2+ movement in DRG, we performed primary sensory neuron soma Ca2+ imaging 1 d after the incision surgery. As in our previous study, using single photon confocal microscopy, we monitored Ca2+ activity and Ca2+ transient response of primary sensory neuron soma in Pirt-GCaMP3 mice with mechanical and thermal stimulation of the paw (Fig. 3). We confirmed that GCaMP3 is expressed in cytosol, shown by the lack of signal in the nucleus, seen as the unstained area in the soma center (Fig. 3A). In this experiment, we defined in vivo “spontaneous Ca2+ activity” as occurring under conditions of no stimulation to the paw or to the animal. Spontaneous Ca2+ activity and Ca2+ oscillation events were detected in a larger number of cell soma in incision mice than in naive mice (Fig. 3B–D, Movies 1, 2). The increased Ca2+ activity of primary sensory soma was reduced in Capsaicin-treated incised mice (q(11) = 4.370, p = 0.0021, Dunnett's test incision vs naive; q(11) = 4.763, p = 0.0011, Dunnett's test incision vs ics-capsaicin; Fig. 3B Movie 3). Spontaneous Ca2+ activity in DRG neuronal cell bodies did not synchronize and were random events (Fig. 3C).
Spontaneous Ca2+ activity of primary sensory neurons in in vivo entire DRG imaging using Pirt-GCaMP3 mice. A, Representative high-resolution images of DRG green fluorescence derived from GCaMP3 in naive, incision, and capsaicin-treated incision mice. B, Total cell numbers exhibiting spontaneous Ca2+ activity in naive, incision, and capsaicin-treated incision mice. C, Representative spontaneous Ca2+ transient responses of primary sensory neurons in incision model. Each arrowhead indicates a spontaneous Ca2+ transient-positive cell. D, Ca2+ transients in spontaneous activity-positive cells of C. Cap-ICS, capsaicin-treated incision. Scale bars: 100 µm. Naive, n = 5; incision, n = 4; Cap-ICS, n = 5. Data represent mean ± SEM; Dunnett's test; *p < 0.05, **p < 0.01, incision versus naive or capsaicin-treated incision.
Related to Figure 3. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure spontaneous activity from naive control.
Related to Figure 3. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure spontaneous activity from incision model.
Related to Figure 3. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure spontaneous activity from capsaicin-treated incision model.
Activation of many primary sensory neurons in the incision model were detected by weak stimulation to the hindpaw plantar skin
The mechanical threshold for paw withdrawal behaviors in the incision animal model was much lower than in naive or capsaicin-treated incision animals (Fig. 1). We, therefore, examined Ca2+ response induced by small and large brush, and by 0.4 and 2.0 g von Frey filament application. Many more cells were activated by small and large brush in the incision model than in naive or capsaicin-treated animals, but this was not seen following 0.4 and 2.0 g von Frey filament stimuli (Fig. 4A,B; Fig. 4B: small brush, q(11) = 4.048, p = 0.0035, Dunnett's test incision vs naive; q(11) = 4.948, p = 0.0008, Dunnett's test incision vs ics-capsaicin; large brush, q(12) = 4.457, p = 0.0015, Dunnett's test incision vs naive; q(12) = 5.835, p = 0.0002, Dunnett's test incision vs ics-capsaicin; 0.4 g von Frey, q(12) = 2.583, p = 0.0433, Dunnett's test incision vs naive; q(12) = 0.2348, p = 0.9606, Dunnett's test incision vs ics-capsaicin; 2 g von Frey, q(11) = 0.4610, p = 0.8548, Dunnett's test incision vs naive; q(11) = 4.149, p = 0.0030, Dunnett's test incision vs ics-capsaicin). No significant differences in Ca2+ transients induced by large brush stimulation were detected among groups (naive, incision, capsaicin-treated incision; Fig. 4C: F(2,1785) = 2.094, p = 0.1235, ANOVA).
In vivo DRG imaging of Ca2+ activity induced by weak mechanical stimulation of primary sensory neurons. A, Representative Ca2+ elevation images induced by large brush stimulation in naive, incision, and capsaicin-treated incision mice. B, Total cell number activated by stimulation with small brush, large brush, 0.4 g von Frey, and 2.0 g von Frey filament in naive, incision, and capsaicin-treated incision mice. C, Ca2+ transient induced by large brush stimulation in naive, incision, and capsaicin-treated incision mice. Black (naive mice), red (incision mice), and blue (capsaicin-treated incision mice) lines indicate average Ca2+ transient. Cap-ICS, capsaicin-treated incision. Scale bars: 100 µm. Naive, n = 5; incision, n = 4–5; Cap-ICS, n = 5. Data represent mean ± SEM. Dunnett's test; *p < 0.05, **p < 0.01, ***p < 0.001, incision versus naive or capsaicin-treated incision mice.
A larger number of cells in primary sensory neurons are activated by mechanical, thermal, and chemical stimuli to the hindpaw
A larger number of cells were activated by 100-g press, 300-g press, 50°C thermal, or 100 mm KCl intraplantar injection in incision animals compared with naive or capsaicin-treated incision animals (Fig. 5A,B, Movies 4, 5, 6, 7, 8, 9, 10, 11, 12; Fig. 5B: 100-g press, q(11) = 3.922, p = 0.0044, Dunnett's test incision vs naive; q(11) = 5.584, p = 0.0003, Dunnett's test incision vs ics-capsaicin; 300-g press, q(11) = 4.925, p = 0.0009, Dunnett's test incision vs naive; q(11) = 8.416, p = 0.0001, Dunnett's test incision vs ics-capsaicin; 50°C, q(11) = 4.669, p = 0.0013, Dunnett's test incision vs naive; q(11) = 7.050, p <0.0001, Dunnett's test incision vs ics-capsaicin; 100 mm KCl, q(10) = 4.196, p = 0.0034, Dunnett's test incision vs naive; q(10) = 6.364, p = 0.0002, Dunnett's test incision vs ics-capsaicin). However, no significant differences in Ca2+ activated cell numbers from acetone application were detected between naive and incision (Fig. 5B: q(11) = 1.866, p = 0.1502, Dunnett's test incision vs naive; q(11) = 3.534, p = 0.0085, Dunnett's test incision vs ics-capsaicin). Ca2+ response induced by 100-g press, 50°C thermal, or 100 mm KCl intraplantar injection were greatly enhanced in the incised animals compared with naive or capsaicin-treated incised animals (Fig. 5C, Movies 4, 5, 6, 7, 8, 9, 10, 11, 12: 100-g press, F(2,7995) = 15.93, p = 0.0001, ANOVA; 50°C, F(2,9060) = 34.26, p = 0.0001; 100 mm KCl, F(2,4935) = 10.71, p = 0.0001, ANOVA).
In vivo DRG imaging of Ca2+ elevation induced by medium to strong stimuli in primary sensory neurons. A, Representative Ca2+ elevation images induced by 100-g press to the hindpaw in naive, incision, and capsaicin-treated incision mice. B, Total cell numbers activated by 100-g press, 300-g press, or hot water to the hindpaw, or acetone to the plantar area, or 100 mm KCl intraplantar injection in naive, incision, and capsaicin-treated incision mice. C, Ca2+ transient induced by 100-g press or 50°C hot water to the hindpaw, or 100 mm KCl intraplantar injection in naive, incision, and capsaicin-treated incision mice. Black (naive mice), red (incision mice), blue (capsaicin-treated incision mice) lines indicate average Ca2+ transients. Cap-ICS, capsaicin-treated incision. Scale bars: 100 µm. Naive, n = 4–5; incision, n = 3–5; Cap-ICS, n = 5. Data represent mean ± SEM. Dunnett's test; *p < 0.05, **p < 0.01, ***p < 0.001 incision versus naive mice; #p < 0.05, ##p < 0.01, ###p < 0.001 incision versus capsaicin-treated incision mice.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 100-g press to the hindpaw from naive control.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 100-g press to the hindpaw from incision model.2+
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 100-g press to the hindpaw from capsaicin-treated incision model.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 50°C water to the hindpaw from naive control.
Related to Figure 5. Representative image of entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 50°C water to the hindpaw from incision model.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 50°C water to the hindpaw from capsaicin-treated incision model.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 100 mm KCl intraplantar injection to the hindpaw from naive control.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 100 mm KCl intraplantar injection to the hindpaw from incision model.
Related to Figure 5. Representative image of in vivo entire L5 DRG neurons GCaMP Ca2+ imaging to measure Ca2+ elevation induced by 100 mm KCl intraplantar injection to the hindpaw from capsaicin-treated incision model.
A larger number of small and medium diameter cells of primary sensory neurons are activated by stimuli to the hind paw in the incision model
We analyzed diameters of primary sensory neurons activated by the stimuli in naive, incision, and capsaicin-treated incision. Cell numbers of small diameter neurons showing spontaneous Ca2+ activity and cell numbers activated by 100-g press, 50°C, and 100 mm KCl intraplantar injection in incised mice were increased compared with naive and capsaicin-treated incised mice (Fig. 6A; spontaneous activity, q(9) = 8.68, p = 0.0001, Dunnett's test incision vs naive; q(9) = 8.50, p = 0.0001, Dunnett's test incision vs ics-capsaicin; 100-g press, q(11) = 4.23, p = 0.0040, Dunnett's test incision vs naive; q(11) = 5.87, p = 0.0005, Dunnett's test incision vs ics-capsaicin; 50°C, q(9) = 4.21, p = 0.0041, Dunnett's test incision vs naive; q(9) = 6.79, p = 0.0002, Dunnett's test incision vs ics-capsaicin; 100 mm KCl, q(8) = 5.03, p = 0.0018 Dunnett's test incision vs naive; q(8) = 9.49, p = 0.0001, Dunnett's test incision vs ics-capsaicin). Cell numbers of medium diameter neurons activated by large brush, small brush, 100-g press, 300-g press, 50°C, and 100 mm KCl intraplantar injection were greatly increased in incised mice compared with naive and capsaicin-treated incised mice (Fig. 6B; large brush, q(10) = 4.44, p = 0.0023 Dunnett's test incision vs naive; q(10) = 5.37, p = 0.0006, Dunnett's test incision vs ics-capsaicin; small brush, q(11) = 3.71, p = 0.0038, Dunnett's test incision vs naive; q(11) = 4.54, p = 0.0020, Dunnett's test incision vs ics-capsaicin; 100-g press, q(9) = 4.27, p = 0.0074, Dunnett's test incision vs naive; q(9) = 6.08, p = 0.0004, Dunnett's test incision vs ics-capsaicin; 300-g press, q(9) = 5.09, p = 0.0012, Dunnett's test incision vs naive; q(9) = 7.54, p = 0.0001, Dunnett's test incision vs ics-capsaicin; 50°C, q(9) = 3.42, p = 0.0135, Dunnett's test incision vs naive; q(9) = 5.60, p = 0.0006, Dunnett's test incision vs ics-capsaicin; 100 mm KCl, q(8) = 2.68, p = 0.0487, Dunnett's test incision vs naive; q(8) = 4.71, p = 0.0028, Dunnett's test incision vs ics-capsaicin). The numbers of large diameter neurons activated by small brush, 100-g press, and 300-g press were also increased in incised animals compared with capsaicin-treated incised mice (Fig. 6C; small brush, q(10) = 3.35, p = 0.0133, Dunnett's test incision vs naive; q(10) = 3.54, p = 0.0099, Dunnett's test incision vs ics-capsaicin; 100-g press, q(9) = 2.92, p = 0.0296, Dunnett's test incision vs naive; q(9) = 3.95, p = 0.0060, Dunnett's test incision vs ics-capsaicin; 300-g press, q(9) = 1.54, p = 0.2547, Dunnett's test incision vs naive; q(9) = 7.54, p = 0.0105, Dunnett's test incision vs ics-capsaicin).
Primary sensory neurons involved in postoperative pain are mainly small and medium diameter. A–F, Analysis of cell diameters of primary sensory neurons activated by stimuli to the hindpaw in naive and incision mice. Cap-ICS, capsaicin-treated incision. Naive, n = 4–5; incision, n = 3–5; Cap-ICS, n = 5. Data represent mean ± SEM. Dunnett's test; *p < 0.05, **p < 0.01, ***p < 0.001 incision versus naive or capsaicin-treated incision mice.
In situ primary sensory nerve fiber Ca2+ imaging in spinal cord sections
We determined that enhanced Ca2+ signaling in the incision model is dependent on the activation of capsaicin-sensitive primary sensory neurons. We examined Ca2+ transients from primary sensory nerve fibers in Lamina I and II in the incision model using spinal cord slices from Pirt-GCaMP3 mice. Ca2+ transients induced by application of 1 μm capsaicin or 50 mm KCl to the ipsilateral side of the incision were significantly enhanced compared with applications to the contralateral side (Fig. 7A–C; Fig. 7A: capsaicin, F(1,252) = 260.8, p = 0.0001, ANOVA ipsilateral vs contralateral; 50 mm KCl, F(1,344) = 10.08, p = 0.0001, ANOVA ipsilateral vs contralateral). However, no significant differences in Ca2+ transients induced by 100 mm KCl were detected between applications to the contralateral and ipsilateral side of the incision (Fig. 7A; 100 mm KCl, F(1,137) = 260.8, p = 0.2400, ANOVA ipsilateral vs contralateral). In capsaicin-treated incision mice, no significant differences were detected between Ca2+ transients induced by applications of capsaicin (1 μm), 50 mm KCl, or 100 mm KCl to the ipsilateral and contralateral side of the incision at any single time point. However, there was a difference in the curves induced by capsaicin (Fig. 7B; capsaicin, F(1,275) = 18.54, p = 0.0001, ANOVA ipsilateral vs contralateral; 50 mm KCl, F(1,298) = 3.635, p = 0.0575, ANOVA ipsilateral vs contralateral; 100 mm KCl, F(1,137) = 0.05, p = 0.8205, ANOVA ipsilateral vs contralateral).
Ca2+ transient imaging of primary sensory neuronal central terminals in in situ sliced spinal cord in the postoperative pain model using Pirt-GCaMP3 mice. A, B, Measurement of Ca2+ transients in in situ sliced spinal cord from vehicle-treated (A) or capsaicin-treated (B) incision Pirt-GCaMP3 mice. Ca2+ transients were induced by bath application of 1 μm capsaicin, 50 mm KCl, or 100 mm KCl. C, Representative Ca2+ transient images of primary sensory neuronal central terminal induced by capsaicin (1 μm) and 50 mm KCl in ipsilateral and contralateral side of sliced spinal cord from vehicle-treated or capsaicin-treated incision mice. White circles show regions of interest (ROIs). Scale bars: 50 µm. Incision mice, n = 4; Capsaicin-treated incision mice, n = 3. Data represent mean ± SEM t test; *p < 0.05, **p < 0.01, ***p < 0.001 ipsilateral versus contralateral.
Discussion
The present study demonstrates that in vivo spontaneous Ca2+ activity at cell bodies of primary sensory neurons, and in vivo Ca2+ responses to mechanical, thermal, and chemical stimuli, are highly enhanced in animals after plantar incision. The Ca2+ response in central terminals of primary sensory neurons is also significantly enhanced in ipsilateral spinal cord of animals after plantar incision. The enhancement of spontaneous Ca2+ activity and Ca2+ response is diminished because of sensory nerve degeneration induced by capsaicin intraplantar injection before surgery. The present study also finds that capsaicin pretreatment alleviates spontaneous, mechanical, and thermal pain by degeneration of skin nerve fibers including TRPV1-positive primary sensory neuron fibers. Our present study renewed clinical interest for wound infiltration of capsaicin to enhance postoperative pain management.
Previous studies have shown that both 1% (200 µl) and 0.05% (200 µl) capsaicin intraplantar injection alleviated spontaneous and thermal pain but not mechanical pain, and did not impact wound healing in a rat incisional pain model (Kang et al., 2010; Tran et al., 2020; Uhelski et al., 2020). The present study used a mouse model plantar incision (Banik et al., 2006; Banik and Brennan, 2009). In our study, 50 µl of 0.05% capsaicin injection into surgical site showed an identical effect; it alleviated spontaneous, mechanical, and thermal pain. In this mouse model, mast cell number was significantly increased in the surgical site, and was mediated by Mas-related G-protein-coupled receptor in response to substance P released by primary sensory nerve fibers in the inflamed skin, contributing to swelling of skin around the wound followed by mechanical and thermal pain (Green et al., 2019). In addition, TRPV1-positive primary sensory neurons express substance P, and TRPV1 ion channels facilitate substance P release in inflammatory conditions (Hsieh et al., 2012; Li et al., 2019). TRPV1 conditional knock-out alleviated thermal pain but not mechanical pain induced by plantar incision. In contrast, our subcutaneous capsaicin injection significantly alleviated mechanical pain. Because TRPV1 was knocked out ubiquitously from early embryogenesis in the previous study, pain behavior phenotypes evoked by primary sensory neuron would likely be different from pain behaviors in our model in which TRPV1-positive nerve degeneration follows intraplantar capsaicin injection. We confirmed that topical application of 100 μm capsaicin to the DRG increased the cell body Ca2+ level of primary sensory neurons (data not shown). Furthermore, piezo channels are top candidates that might play a major role in mechanical pain. Mechanosensitive stretch channel piezo2 is highly expressed in TRPV1-positive neurons (Goswami et al., 2014; Wang et al., 2019) and humans lacking Piezo2 function did not experience allodynia following capsaicin treatment (Fernández-Trillo et al., 2020). TRPV1-positive primary skin nerve degeneration induced by capsaicin pretreatment reduces mechanical pain; as well, Piezo2 is expressed in TRPV1-positive neurons, suggesting a role for Piezo2 in postoperative mechanical pain. It might be possible that species differences between rats and mice in degeneration mechanisms of TRPV1 channel-expressing primary sensory neurons would have an impact on mechanical pain induced by plantar incision.
To assess the effects of intraplantar capsaicin injection onto skin nerve fiber, we visualized primary sensory nerve fibers by immunohistochemistry using PLAP, β-III tubulin, TRPV1 channel, and GFP antibodies. PLAP histochemistry was used to visualize genetically-labeled axon arbors (Wu et al., 2012). Specific expression of PLAP on primary sensory neurons was driven by primary sensory neuron-specific Pirt-cre. β-III tubulin is regarded as a neuronal fiber marker, and is used for detection of primary sensory nerve fiber (Katsetos et al., 1993; Dráberová et al., 1998; Farahani et al., 2019). Around 30−40% of total primary sensory fibers are TRPV1-positive (Bär et al., 2004; Binzen et al., 2006), and capsaicin pretreatment induced degeneration of almost all TRPV1-positive primary sensory nerve fibers. The present results suggest that degeneration of skin nerve fibers induced by capsaicin can alleviate mechanical and thermal pain caused by activation of TRPV1 channels and TRPV1-positive nerve fibers.
In the present study, we assessed spontaneous pain by counting spontaneous foot lifting and conducting an open field test. Foot lifting events associated with incisional pain in animals treated with capsaicin were significantly decreased compared with untreated controls. This result suggests that spontaneous Ca2+ activity of DRG primary sensory neurons induced by TRPV1-positive nerve fibers plays a key role in generation of spontaneous pain. Mediators in DRG and skin, or changes in peri-environmental primary sensory neuron fibers in the skin, could persistently activate TRPV1-positive nerve fibers through activation of TRPV1 channels. Low pH (4.0–5.0) of local skin caused by incision-mediated inflammation will activate TRPV1 and other channels (Tominaga and Tominaga, 2005; Schreml et al., 2010). We also examined spontaneous pain using an open field test. Animals with plantar incision significantly reduced total walking distance and average walking velocity in the open field test. In a previous report, total distance of mouse movement was significantly reduced following chronic constriction injury (Blum et al., 2014) of sciatic nerve-induced neuropathic pain, but was not reduced in a complete Freund's adjuvant (CFA) model of inflammation, or in a spared nerve injury model (Urban et al., 2011). In contrast, significant differences in spontaneous foot lifting were detected in a CFA rat model, but not in a CCI mouse model (Djouhri et al., 2006; Mogil et al., 2010). Thus, as measures of spontaneous pain, spontaneous foot lifting does not necessarily correlate with open field test results. However, in our incisional pain model, spontaneous foot lifting results were consistent with open field test results.
In our previous study, we used Pirt-GCaMP3 mice to monitor in vivo Ca2+ changes in primary sensory neurons in DRG, especially Ca2+ elevation in response to hindpaw stimuli. In the present study, we determined that spontaneous Ca2+ activity of primary sensory neurons in in vivo DRG Ca2+ imaging occurred under conditions of no stimulation to the hindpaw in the incisional pain model. Intraplantar capsaicin injection before the incision abolished enhanced spontaneous Ca2+ activity of DRG primary sensory neurons, and significantly decreased both the number of Ca2+ response cells and Ca2+ transients of each soma caused by plantar stimuli. We conclude that spontaneous Ca2+ activity of DRG primary sensory neurons contributes to spontaneous pain, because intraplantar capsaicin injection significantly reduced both spontaneous pain and spontaneous Ca2+ activity of DRG primary sensory neurons. A previous study has suggested that IGF-2 expression in DRG was increased by plantar incision, but was normalized by the pretreatment of capsaicin injection before the incision (Tran et al., 2020). It is possible that increased IGF-2 resulting from the planar incision evoke spontaneous Ca2+ activity of DRG neurons as an endogenous mediator. IGF-1 and IGF-2 can cross-activate and directly activate both IGF-1 and IGF-2 receptors (Rowzee et al., 2008). Each IGF receptor has different properties in physiological functions (Gary-Bobo et al., 2007; Hakuno and Takahashi, 2018). Additional experiments will be needed to elucidate the mechanisms related to IGF signaling underlying postoperative pain.
In our mouse model of plantar incision, the number of activated cells was significantly increased by stimulation with large brush, 100-g press, 300-g press, 50°C water, or 100 mm KCl, although not by acetone, compared with naive animals and postoperative pain animals treated with intraplantar capsaicin injection. The number of cells activated by a mild stimulus like large brush was much less than numbers activated by harsher stimuli such as 100-g press, 300-g press, and 50°C water. Additionally, we monitored only the surface of DRG using confocal microscopy, and it is possible that the actual cell number activated by mild stimuli like large brush would be greater if we could image deeper into the DRG. Other groups and our own results also showed that the activated cell number is different depending on the stimulus in in vivo DRG and trigeminal ganglia Ca2+ imaging (Kim et al., 2016; Chisholm et al., 2018; Leijon et al., 2019). More detailed analysis reveals that the increased cell number following plantar incision is mainly because of an increase in small to medium diameter cells. These results suggest that plantar incision has specific influences on primary sensory neuron subtypes. The present study shows that TRPV1-positive primary sensory neurons are involved in postoperatively enhanced Ca2+ responses. However, our experiments with capsaicin injection did not indicate which specific primary sensory neuron subtype was involved in each enhanced pain behavior in this model. Other researchers found that primary sensory neurons from TRPV1-cre animals labeled small diameter C fibers and a subset of medium Aδ fibers that innervate Lamina I and II in the spinal cord, especially peptidergic primary sensory neurons. Our present and previous results are consistent with these findings (Bär et al., 2004; Cavanaugh et al., 2011; Mishra et al., 2011; Pogorzala et al., 2013; Le Pichon and Chesler, 2014). Additionally, Mitchell and colleagues found that TRPV1-expressing Aδ fibers are involved in inflammatory pain which is controlled by resiniferatoxin, a strong TRPV1 channel agonist (Mitchell et al., 2010, 2014). Future studies are need to understand specific primary sensory neuron subtypes involved in each enhanced pain behavior using genetic CGRP-cre or MrgprD-cre mouse lines for peptidergic or non-peptidergic fiber positive primary sensory neurons, respectively (Le Pichon and Chesler, 2014).
Like previous studies, our data showed no cold allodynia after plantar incision. We used acetone to assess cold allodynia. Acetone decreases skin temperature to around 15–21°C. The threshold temperature for activation of transient receptor potential melastatin 8 (TRPM8) and transient receptor potential ankyrin 1 (TRPA1) channels is around 25°C and 17°C, respectively (Dhaka et al., 2006; Chen, 2015). Therefore, acetone-induced cooling stimulation in the present study could activate TRPM8 but possibly not TRPA1 channels. Additional experiments are needed to confirm the involvement of TRPA1 channel in incisional pain behaviors.
Some studies showed neuronal plasticity in spinal cord involving increased expression of c-Fos in postoperative pain (Zhu et al., 2006; Guo et al., 2014; Gu et al., 2019; Xu et al., 2019). Only a few studies, using Ca2+ dye or genetically-encoded calcium indicators, have examined the contribution to postoperative pain of Ca2+ activity and Ca2+ signaling in spinal cord. In this study, we monitored Ca2+ activity and Ca2+ signaling at primary sensory neuron central terminals in spinal cord using the Pirt-GCaMP3 mouse model. In each spinal cord slice, we used clearly visible green fluorescence to identify primary sensory nerve fibers in the area of Lamina I and II. Corresponding to in vivo DRG Ca2+ imaging, Ca2+ transients and elevation induced by capsaicin and high KCl (50 mm) in the ipsilateral side were significantly enhanced compared with activity in the contralateral side. The findings suggest that in ddition to increased sensitivity at the site of incision, plantar incision induces plasticity-related changes in primary sensory neuron central terminals in spinal cord. The return of KCl-induced and capsaicin-induced Ca2+ transients' amplitude (ipsilateral spinal cord) to contralateral levels with capsaicin pretreatment suggests that hypersensitivity of central terminals requires intact skin nerve terminals and fibers which probably mediate incision-induced synaptic plasticity at primary sensory neurons.
In summary, in this mouse model of incisional pain, primary sensory neuronal activity is dramatically enhanced, and subcutaneous capsaicin injection before incision surgery causes TRPV1-expressing neuronal nerve fiber degeneration followed by the alleviation of spontaneous, mechanical, and thermal pain, and also significantly reduces Ca2+ elevation and activity of DRG primary sensory neurons. Although capsaicin elevates Ca2+ concentration through the activation of TRPV1 channels, the capsaicin-mediated Ca2+ elevation is weaker than resiniferatoxin-mediated Ca2+ elevation, but strong enough to control TRPV1-mediated pain conditions; resiniferatoxin apparently can control these various TRPV1-related pain types including postoperative pain (Szallasi and Blumberg, 1990; Neubert et al., 2003, 2008; Karai et al., 2004; Hockman et al., 2018; Raithel et al., 2018). Our findings support the use of TRPV1 agonist treatment options for postoperative pain in humans. Previous clinical studies, although limited, have shown an opioid sparing effect of capsaicin instillation on surgical wounds. Instillation of capsaicin into the surgical site reduced opioid use up to 72 h after total knee arthroplasty, despite patients having comparable pain scores (Hartrick et al., 2011; Wiwanitkit, 2012). In a clinical trial investigating the efficacy of a single intraoperative wound instillation of capsaicin, postoperative VAS was reduced during the first 3 d postoperatively, but did not persist beyond this period (Aasvang et al., 2008). Future studies should clarify the efficacy and safety of capsaicin or resiniferatoxin infiltration into surgical sites for postoperative pain management.
Footnotes
This work was supported by the National Institutes of Health Grant R01DE026677 (to Y.K.), University of Texas Health Science Center at San Antonio startup fund (Y.K.), and a University of Texas System STAR Award (Y.K.).
The authors declare no competing financial interests.
- Correspondence should be addressed to Yu Shin Kim at kimy1{at}uthscsa.edu
