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. Author manuscript; available in PMC: 2024 Sep 1.
Published in final edited form as: Neuroscience. 2023 Jul 27;527:92–102. doi: 10.1016/j.neuroscience.2023.07.017

The Utility of Peripherally Restricted Kappa-Opioid Receptor Agonists for Inhibiting Below-Level Pain after Spinal Cord Injury in Mice

Danxu Ma a,†, Qian Huang a,†, Xinyan Gao a, Neil C Ford a, Ruijuan Guo a, Chi Zhang a, Shuguang Liu a, Shao-Qiu He a, Srinivasa N Raja a, Yun Guan a,b
PMCID: PMC10530135  NIHMSID: NIHMS1921185  PMID: 37516437

Abstract

Pain after spinal cord injury (SCI) can be difficult to treat. Drugs that target the opioid receptor (OR) outside the central nervous system (CNS) have gained increasing interest in pain control owing to their low risk of central side effects. Asimadoline and ICI-204448 are believed to be peripherally restricted KOR agonists with limited access to the CNS. This study examined whether they can attenuate pain hypersensitivity in mice subjected to a contusive T10 SCI. Subcutaneous (s.c.) injection of asimadoline (5, 20 mg/kg) and ICI-204448 (1, 10 mg/kg) inhibited heat hypersensitivity at both doses, but only attenuated mechanical hypersensitivity at the high dose. However, the high-dose asimadoline adversely affected animals’ exploratory performance in SCI mice and caused aversion, suggesting CNS drug penetration. In contrast, high-dose ICI-204448 did not impair exploration and remained effective in reducing both mechanical and heat hypersensitivities after SCI. Accordingly, we chose to examine the potential peripheral neuronal mechanism for ICI-204448-induced pain inhibition by conducting in vivo calcium imaging of dorsal root ganglion (DRG) in Pirt-GCaMP6s+/‒ mice. High-dose ICI-204448 (10 mg/kg, s.c.) attenuated the increased fluorescence intensity of lumbar DRG neurons activated by a noxious pinch (400 g) stimulation in SCI mice. In conclusion, systemic administration of ICI-204448 achieved SCI pain inhibition at doses that did not induce notable side effects and attenuated DRG neuronal excitability which may partly contribute to its pain inhibition. These findings suggest that peripherally restricted KOR agonists may be useful for treating SCI pain, but the therapeutic window must be carefully examined.

Keywords: Spinal cord injury, kappa-opioid receptor, pain, dorsal root ganglion, mice

INTRODUCTION

Spinal cord injury (SCI) can lead to debilitating pain that is notoriously difficult to treat (Shiao R and Lee-Kubli CA, 2018). The neuropathic component of SCI pain is broadly classified as above-level, at-level, and below-level with respect to the location of perceived pain in relation to the level of injury (Burke D et al., 2017;Widerstrom-Noga E, 2017;Wieseler J et al., 2010). Most SCIs are contusive in nature and result from incomplete cord damage (Burke D, et al., 2017;Hulsebosch CE et al., 2000;Widerstrom-Noga E, 2017). SCI pain-like behavior also develops after contusive injury in animal models and may share similar fundamental mechanisms with SCI pain in patients (Hulsebosch CE et al., 2009;Hulsebosch CE, et al., 2000;Widerstrom-Noga E, 2017). In our previous studies, a moderate contusive SCI at the T10 vertebral level led to the development of mechanical and heat hypersensitivity in the hind paws of rodents (Duan W et al., 2019;Liu S et al., 2019). The induced sensory hypersensitivity developed at 3–4 weeks post-SCI and persisted for more than 6 weeks, suggesting long-lasting hyperreflexia and hypersensitivity below the level of injury (Duan W, et al., 2019;Liu S, et al., 2019) that may mimic the below-level pain in patients (Hulsebosch CE, et al., 2009;Shiao R and Lee-Kubli CA, 2018;Wieseler J, et al., 2010).

SCI pain is considered a type of central neuropathic pain (Shiao R and Lee-Kubli CA, 2018). However, in addition to etiologies and mechanisms in the central nervous system (CNS), recent studies suggested that the development and maintenance of SCI pain may also be partly attributable to peripheral neuronal mechanisms, such as hyperexcitability and maladaptive changes in dorsal root ganglia (DRG) neurons (Liu S, et al., 2019;Nees TA et al., 2017;Yang Q et al., 2014).

Kappa-opioid receptors (KORs) are widely expressed throughout the brain, spinal cord, and periphery and are associated with Gi/o-coupled signaling (Albert-Vartanian A et al., 2016). KOR agonists have been shown to inhibit voltage-gated calcium channels and exerted analgesic effects comparable to those of mu-opioid receptor (MOR) agonists in some animal pain models (Beck TC et al., 2019;Edwards KA et al., 2018;Naser PV and Kuner R, 2018). Interest has grown in regulating peripheral opioid receptors to reduce pain while avoiding central side effects (Aceves M et al., 2016;Hook MA et al., 2017). Asimadoline (a.k.a. EMD-61753) and ICI-204448 are believed to be peripherally restricted KOR agonists that penetrate the CNS only limitedly after systemic administration (Albert-Vartanian A, et al., 2016;Beck TC and Dix TA, 2019;Beck TC et al., 2019;Caram-Salas NL et al., 2007;Keita H et al., 1995;Negus SS et al., 2012). They may avoid psychoactive side effects known to conventional KOR agonists. Moreover, recent studies have demonstrated the potential utility of some peripherally restricted KOR agonists as non-addictive pain medications (Beck TC and Dix TA, 2019;Beck TC, et al., 2019;Paton KF et al., 2020), such as inhibiting mechanical and heat hypersensitivity in peripheral neuropathic pain and inflammatory pain models (Albert-Vartanian A, et al., 2016;Caram-Salas NL, et al., 2007). However, their potential utility in SCI pain treatment and whether they can exhibit sufficient peripheral selectivity after SCI remain unknown, particularly because SCI may impair the blood-brain barrier (BBB) and thus compromise peripherally restricted drug distribution and action. Therefore, we investigated whether systemic administration of ICI-204448 and asimadoline can reduce established SCI pain in mice and explored the peripheral neuronal mechanisms of drug action in DRG.

EXPERIMENTAL PROCEDURES

Animals

Eight-week-old C57BL/6J mice were purchased from the Jackson Laboratory. As described in our previous studies (Gao X et al., 2020;Kim YS et al., 2016;Liu S, et al., 2019), Pirt-GCaMP6s mice were bred in-house by crossing Pirt-Cre mice with Rosa26-LoxP-STOP-LoxP-GCaMP6s mice, Pirt-GCaMP6s heterozygotes (Pirt-GCaMP6s+/‒) mice express the fluorescent calcium indicator, GCaMP6, in near 90% of all sensory DRG neurons, and not in other peripheral or central tissues, through the Pirt promoter. Both genders of Pirt-GCaMP6s+/‒ mice were used for in vivo calcium imaging of the whole DRG. Our recent study demonstrated inhibitory effects of a peripherally acting MOR-preferring agonist (DALDA) on SCI pain in male mice (Liu S, et al., 2019). To allow data comparison, we also used male mice in current behavioral studies and Western blot analysis. All mice were housed in a temperature-controlled room (24 ± 1°C) with a 12-hour light/dark schedule and free access to both food and water. All experiments were approved by the Johns Hopkins University Animal Care and Use Committee and complied with the National Institutes of Health Guide for the Use of Experimental Animals to ensure minimal animal use and discomfort.

SCI model

SCI was induced as described in our previous study (Liu S, et al., 2019) with a computer-controlled impactor (Impact One™ Stereotaxic Impactor, Leica, Buffalo Grove, IL, USA), which offers four parameters to control the severity of injury: tip diameter, velocity, depth, and dwell time. To avoid a prolonged complete paralysis after severe SCI, which would prevent behavioral testing, we set parameters to produce a moderate contusion SCI in accordance with our previous study (Liu S, et al., 2019): tip diameter was 1.0 mm, velocity was 2.0 m/s, depth was 0.3 mm, and dwell time was 0.1 seconds. Mice were anesthetized with 2% isoflurane delivered through a nose cone, and a partial laminectomy was made at the T10 vertebra, which corresponds to the T12 spinal cord (Fig. 1A). We sutured the muscle layers with 4–0 Polysyn sutures (Angiotech, Reading, PA) and closed the skin with metal clips. Control animals received a sham operation that included skin incision and paravertebral muscle cut at the same level as that used to induce SCI. A prophylactic dose of Enrofloxacin (Sigma-Aldrich, St. Louis, MO, 10 mg/kg, intraperitoneal injection) was administered immediately after surgery and on days 1 and 2 post-surgery to control infection. We performed manual bladder expression twice daily for 3–7 days until reflexive voiding returned.

Fig. 1. Motor dysfunction and mechanical hypersensitivity in hind paws of mice after T10 contusive spinal cord injury (SCI).

Fig. 1.

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(A) Schematic diagram illustrating a contusion at the T10 vertebrate level to create a moderate SCI model. Mechanical stimulation with von Frey filaments was applied to the mid-plantar area of both hind paws. (B) Changes in Basso mouse scale (BMS) scores from day 1–42 post-SCI (n = 20) and sham operation control (skin incision, n = 10, F(1,28)=17.16, P <0.001). (D) Changes in paw withdrawal frequencies to low force (0.07 g, F(1,28)=12.68, P <0.01) and high force (0.4 g, F(1,28)=8.01, P <0.01) mechanical stimulation from day 7–42 in SCI (n = 20) and control groups (n = 10). (E) Changes in paw withdrawal latencies to noxious heat stimulation (Hargreave’s test) from day 7–42 post-SCI (n = 20) and sham operation (n = 10, F(1,28)=3.58, P =0.07). B-E: Data from both hind paws were averaged in each animal for analysis. Two-Way mixed model ANOVA with Bonferroni post hoc test. Data are expressed as mean ± SD. *P<0.05, **P<0.01 and ***P<0.001 versus control; #P<0.05, ##P<0.01 and ###P<0.001 versus day 0 (pre-injury).

Behavioral tests

Locomotor activity assay

The Basso Mouse Scale (BMS) score was used to evaluate locomotor activity after SCI. Testing was carried out as described previously (Basso DM et al., 2006;Liu S, et al., 2019). Briefly, animals were placed in an open field (72 × 72 cm) and allowed to explore freely for 5 minutes, after which hind-limb movements were scored according to the BMS. Scores range from 0, indicating no hind-limb movement, to 9, indicating normal, coordinated gait. Changes in motor function were evaluated by comparing the BMS score at day 0 (before surgery) to that at days 1, 3, and 7 post-surgery and continuing weekly for 6 weeks. Data from both hind limbs were averaged for analysis.

Mechanical pain test

Mechanical testing was carried out as described in our previous studies (Guan Y et al., 2010;Liu S, et al., 2019). A mouse was placed on an elevated mesh screen and a calibrated von Frey filament was applied to one hind paw for approximately 1 second with 1–2 second intervals. This stimulation was repeated 10 times. Both hind paws were tested with a 5-minute intervening break. Each hind paw received two stimulation strengths (low force: 0.07 g; high force: 0.4 g). We scored paw withdrawal manually to yield the paw withdrawal frequency (PWF) for each block of 10 trials. PWF is expressed as a percent response frequency. Data from both hind paws were averaged for analysis.

Heat pain test

Thermal hypersensitivity was tested as described previously (Hargreaves K et al., 1988;Liu S, et al., 2019). Individual mice were placed on the glass platform of a Hargreaves device (IITC model 390, Woodland Hills, CA) under a plastic box (4.5 × 5 × 10 cm) to habituate for at least 30 minutes. A thermal radiant stimulus was positioned beneath one hind paw until the paw was withdrawn. We applied a cutoff time of 30 seconds to prevent tissue injury. Paw withdrawal latency (PWL) was measured automatically. Each hind paw was measured 3 times, with a 5-minute interval between trials. Mechanical and heat sensitivity tests were performed before surgery (day 0) and weekly from 1 to 6 weeks after surgery. Data from both hind paws were averaged for analysis.

Open field test

Spontaneous exploration was measured as an indicator of locomotor activity. Mice were placed in an open field chamber (40 × 40 × 33 cm) for 10 minutes, and their movements were recorded with an automated video tracking system (Panlab SMART 3.0 software, Harvard Apparatus, Barcelona, Spain). Locomotor activity was then estimated from the total distance travelled and ratio of time spent in the center of the field.

Conditioned place aversion (CPA)

The apparatus consisted of a three-chambered box, each chamber with a unique set of visual and tactile cues. The task spanned 4 days. On day 1, mice were allowed to habituate to the full apparatus for 30 minutes. On day 2, pre-conditioning day, mice were recorded as they freely explored all three chambers for 15 minutes. On day 3, mice received vehicle treatment and were restricted to either the left or the right chamber for 1 hour after injection; 4 hours later mice received drug treatment and were restricted to the opposite chamber for 1 hour after injection. Chambers were counterbalanced across all mice. On day 4, post-conditioning day, mice were again recorded as they freely explored all three chambers for 15 minutes. This conditioning protocol has been validated in our previous studies (Li Z et al., 2017;Liu S, et al., 2019;Tiwari V et al., 2018). Preference or aversion for the drug treatment was calculated as an increase or decrease, respectively, in time spent in the drug-paired chamber on post-conditioning day as compared to that on the pre-conditioning day. Animals that displayed a strong preference for one chamber on the pre-conditioning day (spending more than 80% of the total time in a single chamber) were excluded from further analysis.

In vivo calcium imaging

Pirt-GCaMP6s+/‒ of both sexes were used for in vivo calcium imaging study as described in our previous studies (Gao X, et al., 2020;Liu S, et al., 2019;Tiwari V, et al., 2018). At 6 weeks post-SCI, spontaneously breathing mice were anesthetized with 1.5% isoflurane. The right leg and the back were shaved, and the L4 DRG was exposed. Mice were then positioned under a laser scanning confocal microscope (Leica TCS-SP8, Wetzlar, Germany) via micromanipulator-mounted forceps clamped to the spinal column at L2 and L6. Throughout the imaging procedure, 1.5% isoflurane in O2 was used to maintain anesthesia, and a homeothermic blanket system was used to maintain temperature.

Images were acquired with an EM-CCD camera equipped with a 0.5 N.A. macro dry objective. Five image z-stacks (spanning 200–300 μm of tissue depth) were collected in a time series for each stimulus. Before and 1 hour after vehicle (10% DMSO in saline, s.c.) or ICI-204448 (10 mg/kg, s.c.) was subcutaneously administered to each mouse, the right paw was stimulated with a 400 g force mechanical pinch stimulation. In each experiment, the experimenter was blind to drug treatment condition. The pinch (10 seconds) was applied with a calibrated mouse pincher system (2450, IITC Rodent Pincher, IITC Life Science, Inc.) with a tip of 0.8 mm diameter. The force (g) applied during stimulation was displayed on the transducer.

An investigator blind to drug treatment conditions screened the image time series to identify cells that increased in fluorescence with stimulation; these cells were marked for further analysis. Changes in fluorescence intensity were quantified for each cell in each frame of a time series by calculating the fluorescence in that frame (F) relative to the starting (pre-stimulation) frame (F0). Cells were defined as being activated by stimulation when the increase of evoked signal amplitude was at least 0.2-fold of F0, i.e. F/F0 ≥ 1.2 fold (120%) (Gao Q et al., 2020;Liu S, et al., 2019). We compared the fluorescence intensity (F/F0) of all the surveyed neurons responding to each test stimulus before and after drug treatment. Small, medium, and large neurons were defined as having somal areas of <450 μm2, 450–700 μm2, and >700 μm2, respectively.

Western blotting

The DRG and sciatic nerve tissues were homogenized in lysis buffer on ice. Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were first blocked in 5% non-fat dry milk in Tris-buffered saline, and then incubated overnight with mouse-anti-KOR primary antibody (1:300; Santa Cruz Biotechnology, Dallas, TX) at 4°C. Membranes were washed 3 times with Tris-buffered saline containing Tween-20, and then incubated for 2 hours with horseradish peroxidase–conjugated goat anti-mouse secondary antibody (1:10,000; Jackson ImmunoResearch, West Grove, PA) at room temperature. GAPDH (1:100,000; Millipore, Germany) was used as a loading control. The intensity values of target bands were measured by ImageJ (Image J 1.50i, NIH).

Drug preparation

ICI-204448 was purchased from Tocris Bioscience (Minneapolis, MN), and asimadoline was obtained from MilliporeSigma (Germany). Based on previous studies (Caram-Salas NL, et al., 2007;Snyder LM et al., 2018), as well as our findings in a pilot study, we used doses of 1 and 10 mg/kg for ICI-204448, and 5 and 20 mg/kg for asimadoline in animal behavior studies. All drugs were dissolved in vehicle (10% DMSO in saline) as instructed by the manufacturers. Experimenters who conducted behavioral tests were blinded to drug treatment.

Data analysis and statistics

Data were analyzed with GraphPad Prism 7 software, and results are expressed as mean ± standard deviation (SD) or medians (interquartile range). Two-way analysis of variance (ANOVA), followed by Bonferroni correction, was used to compare treatment effects in behavioral experiments (mechanical, heat, open field tests and CPA), data from both hind limbs were averaged for analysis. For western blot analysis, data were compared by using Student’s t-test. Data that were normally distributed were analyzed using a two-way analysis of variance (ANOVA). For data that did not meet the basic assumptions for parametric testing, paired nonparametric statistics (Wilcoxon matched-pairs signed rank test) were used. Statistical significance was defined as P < 0.05.

RESULTS

Moderate contusive SCI induced transient motor dysfunction but prolonged sensory hypersensitivity in the hind paws

Mice exhibited significant reductions in BMS scores beginning on the first day after SCI. Scores remained significantly lower than those of controls for 2 weeks (n = 20, Fig. 1B), but returned to near pre-surgery levels at 3 weeks post-SCI, indicating significant motor function impairments only during the early phase after SCI. Sham-operated mice (control) showed no significant change in BMS score from baseline (n = 10).

Mechanical and heat hypersensitivity in both hind paws were measured weekly after surgery as indications of below-level sensory function (Fig. 1C,E). Compared to pre-injury, the PWFs to high-force (0.4 g) mechanical stimulation were significantly reduced at 1-week post-SCI, but not after sham operation. The reduction may have been due to severely impaired motor function during the early post-SCI time period. However, PWFs increased significantly and remained at a peak level from 4 to 6 weeks after SCI (Fig. 1C). PWLs to radiant heat stimulation at the hind paws decreased at 4- and 6-weeks post-SCI (n = 20) compared to pre-injury levels (Fig. 1D). Sham-operated mice showed no significant change from baseline in either PWF or PWL.

Changes in KOR expression in the peripheral nervous system were examined at 6 weeks after SCI or sham operation, the time point when drug tests were conducted. Total KOR protein expressions in the lumbar DRGs (L3–5, Fig. 2A, Suppl. Fig. 1A) and the sciatic nerves (Fig. 2B, Suppl. Fig. 1B) were comparable between SCI (n = 7) and sham-operated mice (n = 4).

Fig. 2. SCI did not change KOR protein expression in lumbar DRGs and sciatic nerves.

Fig. 2.

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(A) Representative immunoblots and quantification of KOR protein level in bilateral lumbar (L3–5) dorsal root ganglions (DRGs) in SCI mice (S, n = 7) and sham-operated controls (C, n = 4). Tissues from the left and right sides were harvested at day 42 post-SCI or sham-operation. (B) Representative immunoblots and quantification of KOR protein level in bilateral sciatic nerves in SCI (n = 7) and sham-operated mice (n = 4). GAPDH served as an internal control. Data are expressed as mean ± SD. Unpaired student’s t-test.

Systemic administration of asimadoline reduced mechanical and heat hypersensitivities but also induced side effects in SCI mice

Mice that showed significant hypersensitivity to mechanical and heat stimulation in hind paws at 6 weeks post-SCI were selected for drug testing. Asimadoline (Fig. 3), ICI-204448 (Fig. 4), or vehicle (10% DMSO in saline), was injected subcutaneously (s.c.) into the back of awake animals.

Fig. 3. Systemic administration of asimodaline inhibited mechanical and heat hypersensitivity in SCI mice but also impaired locomotor function at the high dose.

Fig. 3.

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(A) Changes of paw withdrawal frequencies to low force (0.07g) and high force (0.4 g) mechanical stimulation after subcutaneous (s.c.) injection of asimodaline (5, 20 mg/kg, n = 12/dose) or vehicle (10% DMSO in saline, n = 24) at day 42 post-SCI. (B) Changes in paw withdraw latency to radiant heat stimulation after asimodaline (n = 12 /dose) or vehicle treatment (n = 24). (C) The total distance traveled in 10 minutes at 1 hour after injection of asimodaline (5, 20 mg/kg, s.c., n = 12/dose) or vehicle (n = 24) in SCI mice. (D) The percentage of time spent in the center zone at 60 minutes after asimodaline or vehicle treatment. A-B: Data from both hind paws were averaged in each animal for analysis. Two-Way mixed model ANOVA with Bonferroni post hoc test. Data are expressed as mean ± SD. **P<0.01 and ***P<0.001 versus vehicle; ##P<0.01 and ###P<0.001 versus pre-drug.

Fig. 4. ICI-204448 inhibited mechanical and heat hypersensitivity in SCI mice at doses without impairing locomotor function.

Fig. 4.

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(A) Changes in paw withdrawal frequencies to low force (0.07g) and high force (0.4 g) mechanical stimulation after subcutaneous (s.c.) injection of ICI-204448 (1, 10 mg/kg, n = 12/dose) or vehicle (10% DMSO, n = 24) at day 42 post-SCI. (B) Changes in paw withdrawal latency to heat stimulation after ICI-204448 (1, 10 mg/kg, s.c., n = 12/dose) or vehicle (10% DMSO, n = 24) in SCI mice. (C) Examples of exploration activity of SCI mice in the open field test (10 minutes) before and 1 hour after injection of ICI-204448 or vehicle. (D) The total distance traveled in 10 minutes at 1 hour after injection of ICI-204448 (1, 10 mg/kg, s.c., n = 12 /dose) or vehicle (n = 24). (D) The percentage of time spent in the center zone 1 hour after ICI-204448 or vehicle. A-B: Data from bilateral hind paws were averaged in each animal for analysis. Two-Way mixed model ANOVA with Bonferroni post hoc test. Data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 versus vehicle; #P<0.05, ###P<0.001 versus pre-drug.

High-dose asimadoline (20 mg/kg, s.c., n = 12) significantly decreased PWF to both low-force and high-force mechanical stimulation and increased PWL to heat stimulation in SCI mice 1 hour after injection (Fig. 3A,B). Low-dose asimadoline (5 mg/kg, s.c., n = 12) only significantly increased heat PWL (Fig. 3B).

In the open field test, locomotor activity was indicated by the total distance traveled, and exploration activity was assessed as the percent time spent in the center zone. Subcutaneous injection of high-dose asimadoline (20 mg/kg, s.c., n = 12) significantly reduced total distance traveled at 1 hour post-drug (Fig. 3C) compared to pre-drug level and that of vehicle-treated SCI mice, suggesting impaired locomotor function and exploratory performance after drug. The time that SCI mice spent in the center zone was not significantly changed in the open field test (Fig. 3D).

Because the impaired locomotor function by high-dose asimadoline may be due to CNS drug penetration after SCI, we carried out CPA test to examine if high-dose asimadoline induces aversion, one of the characteristic side effects of centrally acting KOR agonists (Chefer VI et al., 2013;Ehrich JM et al., 2015). In the CPA test, while naïve mice (n = 9, Suppl. Fig. 2A) spent similar amounts of time in chambers paired with high-dose asimadoline (20 mg/kg, s.c.) after drug conditioning, SCI mice (n = 10, Suppl. Fig. 2B) spent significantly less time in the asimadoline-paired chamber compared to vehicle-paired chamber, indicating a drug-induced aversion.

Systemic administration of ICI-204448 inhibited mechanical and heat hypersensitivities in SCI mice without inducing notable side effects

High-dose ICI-204448 (10 mg/kg, s.c., n = 12) significantly decreased PWF to high-force mechanical stimulation and increased PWL to heat stimulation in SCI mice 1 hour after injection (Fig. 4A,B), as compared to values at pre-drug baseline and in vehicle-treated group (n = 24). Low-dose ICI-204448 (1 mg/kg, s.c., n = 12) only significantly increased PWLs (Fig. 4B). In the open field test, neither dose of ICI-204448 (n = 12/dose) changed the total distance traveled (Fig. 4C, D) or the time that animal spent in the center zone at 1 hour after injection (Fig. 4E).

ICI-204448 attenuated DRG neuronal calcium responses to noxious pinch stimulation in SCI mice

Neurons exhibit calcium-based excitability, and increased intracellular calcium transients in response to internal and external stimulation may serve as an alternative indicator of cell activity. This can be monitored using genetically encoded calcium indicators (GECIs) such as GCaMP (Iseppon F et al., 2022). Because high-dose ICI-204448 (10 mg/kg, s.c.) reduced both mechanical and heat hypersensitivities without impairing animals’ exploratory performance in SCI mice, we conducted in vivo calcium imaging of DRG in Pirt-GCaMP6s+/‒ mice to examine the peripheral neuronal mechanisms for ICI-204448 to inhibit mechanical pain (Fig. 5A,B). ICI-204448 (10 mg/kg, s.c.) did not decrease the number of small, medium and large neurons (calculated as a percentage of all neurons surveyed in each subpopulation) activated by hind paw pinch stimulation (400 g) 1 hour after injection in SCI mice, as compared to those activated before drug treatment (post- vs pre-injection: small, 365 vs 442; medium, 224 vs 242; large, 154 vs 166 in a total of 1233 neurons surveyed from 8 mice; Fig. 5C). Vehicle injection also did not change the percentage of DRG neurons that responded to pinch stimulation (post- vs pre-injection: small, 297 vs 287; medium, 228 vs 232; large, 144 vs 153 in a total of 856 neurons surveyed from 5 mice; Fig. 5C). However, further analysis showed that ICI-204448, but not vehicle, significantly reduced the fluorescence intensity (F/F0) of neurons responding to pinch stimulation in each subpopulation 1 hour after injection, as compared to that evoked before drug administration (Fig. 5D).

Fig. 5. ICI-204448 attenuated calcium responses of lumbar DRG neurons to mechanical pinch stimulation in Pirt-GCaMP6s+/‒ mice at 6 weeks after SCI.

Fig. 5.

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(A) Schematic diagram illustrates the setup for in vivo calcium imaging of L4 DRG with a confocal microscope (Leica SP8) in anesthetized Pirt-GCaMP6s+/‒ mice. (B) Left: Representative images show increased fluorescence intensity in DRG neurons after mechanical pinch (400 g) stimulation at the hind paw before and at 1 hour after ICI-204448 (10 mg/kg, s.c.) injection. Right: A high-magnification image of the boxed area in panel B with examples of three activated cells marked with different colored arrows. Examples of heat map of responses of these neurons responding to the stimulation. (C) Proportions of small, medium and large DRG neurons that were activated (F/F0 > 120%) by pinch stimulation among all neurons surveyed in each subpopulation before and at 1 hour after vehicle (10% DMSO, n = 5) and ICI-204448 (10 mg/kg, s.c., n = 8) treatment at 6 weeks post-SCI. DRG neurons were categorized into three subpopulations according to somal areas as follows: <450 μm2 (small), 450–700 μm2 (medium), and >700 μm2 (large), respectively. Data are expressed as mean ± SD. Two-Way mixed model ANOVA with Bonferroni post hoc test. (D) Quantification of fluorescence intensity of small, medium, and large DRG neurons activated by hind-paw pinch stimulation before and after vehicle (n = 5 mice) or ICI-204448 treatment (n = 8 mice) in SCI mice. The evoked fluorescence intensities of small, medium, and large neurons by pinch stimulation were significantly decreased after ICI-204448 treatment, as compared to pre-injection. Wilcoxon matched-pairs signed rank test. Data are expressed as medians (interquartile range). *P < 0.05, **P < 0.01, ***P<0.001 versus pre-injection.

DISCUSSION

Pain is a common and debilitating complication of SCI that can significantly impair quality of life. Our study showed that subcutaneous administration of ICI-204448 and asimadoline, believed to be peripherally acting KOR agonists (Albert-Vartanian A, et al., 2016;Negus SS, et al., 2012), attenuated both mechanical and heat hypersensitivities in SCI mice. However, high-dose asimadoline impaired exploratory behavior and tended to induce aversion in SCI mice. In contrast, ICI-204448 did not cause notable side effects at doses effective for pain inhibition. Mechanistically, the suppression of DRG neuron excitability may partly contribute to ICI-204448-induced mechanical pain inhibition in SCI mice.

KORs are expressed throughout the nervous system, including the brain, spinal cord, DRG (Cunha TM et al., 2012;Gross RA and Macdonald RL, 1987;Moy JK et al., 2020), and peripheral nerves (Albert-Vartanian A, et al., 2016;Caram-Salas NL, et al., 2007;Keita H, et al., 1995;Snyder LM, et al., 2018). Immunohistochemistry and in situ hybridization studies have shown that KOR is expressed in small, medium, and large DRG neurons (Gross RA and Macdonald RL, 1987;Ji RR et al., 1995;Moy JK, et al., 2020;Snyder LM, et al., 2018). Using KOR-cre knock-in allele and viral tracing, a recent study identified functional KORs in both peptidergic DRG neurons and low-threshold mechanoreceptors in mice. KOR mRNA was also detected in peptidergic DRG neurons in humans (Snyder LM, et al., 2018). Our western blot analysis revealed no significant change in KOR expression in lumbar DRGs and sciatic nerves at 6 weeks after SCI. These findings differ from MOR expression, which increased in sciatic nerves after SCI (Liu S, et al., 2019). We speculated that expression of opioid receptor subtypes might be differentially regulated by genetic and epigenetic mechanisms after SCI.

Activation of KORs can induce membrane hyperpolarization by activating G-protein–gated inwardly rectifying potassium channels, and decrease neuronal excitability and presynaptic neurotransmitter release by inhibiting voltage-gated calcium channels (Grudt TJ and Williams JT, 1993;Snyder LM, et al., 2018). This could contribute to pain inhibition. Local injection of U50488, a selective KOR agonist, also blocked PGE2-induced mechanical hypersensitivity by stimulating the PI3Kγ/AKT/nNOS/NO pathway in DRG neurons (Cunha TM, et al., 2012). KOR signaling at peripheral nerve terminals can inhibit neurogenic inflammation and nociceptor sensitization (Snyder LM, et al., 2018). Here, our in vivo GCaMP6s imaging of whole DRG, a new technology that has led to many advances in our understanding of somatosensation in the nervous system (Anderson M et al., 2018;Gao X, et al., 2020;Miller RE et al., 2018), showed that ICI-204448 (10 mg/kg, s.c.) attenuated evoked calcium responses of DRG neurons to mechanical stimulation in Pirt-GCaMP6s+/‒ mice. This suggests that attenuation of DRG neuronal excitability by ICI-204448 may contribute, in part, to its inhibition of mechanical pain hypersensitivity after SCI. However, calcium transients are not as sensitive and fast as action potentials in reflecting changes in neuronal activities, so the in vivo effects of ICI-204448 on DRG neuron activity need to be confirmed through electrophysiology recording. It remains possible that other sites and mechanisms may also contribute to ICI-204448-induced pain inhibition in SCI mice.

In contrast to ICI-204448, which reduced both mechanical and heat hypersensitivities in SCI mice, subcutaneous injection of dermorphin [D-Arg2, Lys4] (1–4) amide (DALDA), a peripherally acting MOR-preferring agonist (Tiwari V, et al., 2018;Tiwari V et al., 2016), only inhibited heat hypersensitivity, but could not inhibit mechanical hypersensitivity after SCI even at 10 mg/kg (Liu S, et al., 2019). Mechanical and heat hypersensitivities involve different cellular and molecular mechanisms, and may require different treatment strategies. Peripherally restricted agonists to MOR and KOR may possess different utilities in reducing different pain modalities after SCI.

Activation of KORs in the brain causes many side effects such as aversion and sedation, dysphoria, hallucinations, and psychosis (Beck TC, et al., 2019;Chefer VI, et al., 2013;Ehrich JM, et al., 2015). Aversion is a characteristic drug action of centrally acting KOR agonists (Chefer VI, et al., 2013;Ehrich JM, et al., 2015), such as U-50488, which produces dose-related place aversion after systemic drug administration. An important mechanism implicated in this drug action is the modulation of mesolimbic dopamine circuitry such as by reducing dopamine release (Beck TC, et al., 2019;Chefer VI, et al., 2013;Ehrich JM, et al., 2015;Navratilova E et al., 2019). Because the endothelium of vessels supplying DRGs lacks tight junctions (Abram SE et al., 2006;Devor M, 1999), drugs can directly affect neuronal soma and change their excitability in DRG without hindrance from the blood-nerve barrier. Previous studies suggested that both ICI-204448 and asimadoline poorly penetrate the CNS after systemic administration (Albert-Vartanian A, et al., 2016;Caram-Salas NL, et al., 2007;Keita H, et al., 1995;Negus SS, et al., 2012). Therefore, systemic administration of peripherally restricted KOR agonists may reduce pain by activating KORs in the DRG and peripheral nerves, and theoretically, spare aforementioned central side effects. Here, although asimadoline did not induce aversion after drug conditioning in naïve mice, the high-dose asimadoline induced aversion and significantly impaired exploratory behavior in SCI mice, suggesting CNS drug penetration after SCI. Since the procedure to induce SCI disrupts the blood-spinal cord barrier, CNS drug penetration could be partly due to leakage of the blood-spinal cord barrier or BBB in SCI mice (Abram SE, et al., 2006;Naser PV and Kuner R, 2018). Additionally, it may also result from changes in drug pharmacokinetics or neuronal function in mesolimbic dopamine circuitry after SCI (Kuiper H et al., 2021). Due to potential CNS drug penetration causing aversion and sedation, we were unable to use operant behavior tests, such as CPP, to examine whether peripherally acting KOR agonists can inhibit spontaneous and ongoing pain after SCI, which warrants further study.

It remains unclear why side effects were observed only with high-dose asimadoline, but not ICI-204448 after SCI. Asimadoline contains a hydrophobic diphenylmethyl group combined with a hydrophilic hydroxyl group, which may hinder its diffusion across the membranes of the BBB (Albert-Vartanian A, et al., 2016). However, previous studies showed that asimadoline failed to show clinically relevant efficacy at doses that lacked CNS adverse effects (Albert-Vartanian A, et al., 2016;Vanderah TW et al., 2008), which is in line with current findings. In contrast, ICI-204448 is a hydrophilic and non-quaternary derivative that may have lower CNS penetrability than asimadoline (Rawls SM et al., 2005;Snyder LM, et al., 2018). However, it remains possible that ICI-204448 may also induce central side effects if the dose is further increased. Although subcutaneous injection of ICI-204448 at 10 mg/kg did not cause notable side effects in the current study, it induced sedative effects at 2 mg/kg after intravenous injection (Beck TC, et al., 2019;Caram-Salas NL, et al., 2007;Keita H, et al., 1995). Intriguingly, one KOR agonist, U50488, did not inhibit basal adenylyl cyclase activity, but inhibited adenylyl cyclase and thermal allodynia after pretreatment with bradykinin, suggesting an increased drug efficacy after inflammation (Berg KA et al., 2011). Accordingly, the efficacy, safety profile, and amount of CNS drug penetration of different peripherally restricted KOR agonists after SCI are not only dose-related but also affected by chemical properties, pharmacokinetics and pharmacodynamics of the drug, route of administration, integrity of BBB, and animal conditions.

Drugs that target the opioid receptors outside the CNS may offer potential as lower-risk analgesics. Our findings suggest that certain peripherally restricted KOR agonists may reduce SCI pain, partly by inhibiting DRG neuron excitability. Avoiding central adverse effects by targeting KORs in PNS could present a major improvement over conventional medications for treating SCI pain. Some newly-developed peripherally restricted KOR agonists, such as ZYKR1 (Jain MR et al., 2022), CR845 and HSK21542 (Wang X et al., 2021) have shown promising efficacy for attenuating pain and pruritus. In particular, CR845 has completed phase III clinical trials and received FDA approval for the treatment of pruritis in adults undergoing haemodialysis (Fishbane S et al., 2020;Fugal J and Serpa SM, 2023;Lipman ZM and Yosipovitch G, 2020). However, it remains important to determine if these drugs can achieve sufficient pain inhibitory efficacy at doses that lack CNS adverse effects after SCI. The debilitating nature of SCI pain and the lack of efficient treatments justify developing new peripherally acting KOR agonists and studying their therapeutic window in SCI pain treatment.

Supplementary Material

1

HIGHLIGHTS.

  • ICI-204448 and asimadoline are believed to act as peripherally acting kappa-opioid receptor agonists.

  • Systemic administration of ICI-204448 and asimadoline reduced below-level pain hypersensitivity in SCI mice.

  • However, high-dose asimadoline adversely affected the exploratory performance of SCI mice and caused aversion.

  • Systemic administration of ICI-204448 also attenuated calcium responses in DRG neurons of SCI mice.

  • While peripherally restricted kappa agonists may help reduce SCI pain, the therapeutic window must be carefully examined.

Acknowledgments:

The authors thank Claire F. Levine, MS (scientific editor, Department of Anesthesiology/CCM, Johns Hopkins University) for editing the manuscript. The authors thank Dr. Xinzhong Dong (professor, the Solomon H. Snyder Department of Neuroscience, Johns Hopkins University) for providing Pirt-Cre mice.

Funding sources:

This study was conducted at Johns Hopkins University. This study was supported by a seed grant (Y.G.) from the Neurosurgery Pain Research Institute at the Johns Hopkins University and was subsidized by National Institutes of Health (Bethesda, Maryland, USA) grants NS110598 (Y.G.), NS026363 (S.R.), and NS117761 (Y.G.). Funders had no role in study design, data collection, data interpretation, or in the decision to submit the work for publication. This work was facilitated by the Pain Research Core funded by the Blaustein Fund and the Neurosurgery Pain Research Institute at Johns Hopkins University.

Footnotes

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Conflict of interest: None of the authors has a commercial interest in the material presented in this paper. There are no other relationships that might lead to a conflict of interest in the current study.

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