Abstract
The development of painful paclitaxel-induced peripheral neuropathy (PIPN) represents a major dose-limiting side effect of paclitaxel chemotherapy. Here we report a promising effect of duvelisib (Copiktra), a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, in preventing paclitaxel-induced pain-like behavior and pronociceptive signaling in DRGs and spinal cord dorsal horn (SCDH) in rat and mouse model of PIPN. Duvelisib blocked the development of mechanical hyperalgesia in both males and females. Moreover, duvelisib prevented paclitaxel-induced sensitization of TRPV1 receptors, and increased PI3K/Akt signaling in small-diameter DRG neurons and an increase of CD68+ cells within DRGs. Specific optogenetic stimulation of inhibitory neurons combined with patch-clamp recording revealed that duvelisib inhibited paclitaxel-induced weakening of inhibitory, mainly glycinergic control on SCDH excitatory neurons. Enhanced excitatory and reduced inhibitory neurotransmission in the SCDH following PIPN was also alleviated by duvelisib application. In summary, duvelisib showed a promising ability to prevent neuropathic pain in PIPN. The potential use of our findings in human medicine may be augmented by the fact that duvelisib is an FDA-approved drug with known side effects.
SIGNIFICANCE STATEMENT We show that duvelisib, a novel FDA-approved PI3Kδ/γ isoform-specific inhibitor, prevents the development of paclitaxel-induced pain-like behavior in males and females and prevents pronociceptive signaling in DRGs and spinal cord dorsal horn in rat and mouse model of paclitaxel-induced peripheral neuropathy.
Introduction
A fine balance between excitatory and inhibitory neurotransmission in the spinal cord dorsal horn (SCDH) is essential for normal nociceptive and somatosensory processing. Its disruption can lead to the development of pathologic pain (Zeilhofer et al., 2012). Impairments of both excitatory and inhibitory synaptic transmission at the spinal cord level have been shown to contribute to pain development as a side effect of paclitaxel (PAC) chemotherapy (Chen et al., 2014; Li et al., 2015; Yadav et al., 2015).
PAC chemotherapy is widely used in clinical practice for the management of solid tumor cancers. Unfortunately, the therapy is often limited by PAC-induced peripheral neuropathy (PIPN), a chronic painful condition associated with neuropathic pain development that is frequently resistant to standard analgesics and significantly affects the patient's quality of life (Boyette-Davis et al., 2018). PIPN is also associated with neuroinflammation in the CNS and PNS, resulting in sensitization of spinal nociceptive processing (Kalynovska et al., 2020; Meesawatsom et al., 2020).
Phosphatidylinositol 3-kinases (PI3Ks) are key signaling molecules regulating cellular survival, proliferation, differentiation, and metabolism. A growing body of evidence also reports that PI3K signaling within the nervous system, notably in the spinal cord, DRGs, and peripheral nerves, contributes to nociceptive signaling and the development of pain-like behavior in animal models. The role of PI3Ks in nociceptive signaling was reported under inflammatory conditions (Zhuang et al., 2004; Pezet et al., 2008; Wigerblad et al., 2017), after nerve injury (Xu et al., 2007; Liu et al., 2018), in bone cancer-induced pain (Fang et al., 2015; Zhang et al., 2019), in plantar incision-induced postoperative pain (Xu et al., 2019), in myofascial pain (Zhang et al., 2020), as well as in PIPN (Adamek et al., 2019; Manjavachi et al., 2019; Huang et al., 2020). Inhibition of the PI3K signaling in all of these studies alleviated pain-like behavior and/or reduced pro-inflammatory and pro-nociceptive signaling. PI3K was shown to be involved in the regulation of membrane trafficking of several receptors and ion channels within DRG and spinal cord neurons and thus may contribute to peripheral/central sensitization. This was observed for glutamatergic AMPARs (Wigerblad et al., 2017), several types of voltage-gated Ca2+ channels (Viard et al., 2004), acid-sensing ion channel 1 (Duan et al., 2012), and transient receptor potential vanilloid 1 (TRPV1) (Zhang et al., 2005; Stein et al., 2006).
PI3Ks family is divided into three classes (I, II, and III) according to structural and functional aspects. Class I is best understood and further divided into Class IA (PI3Kα, PI3Kβ, and PI3Kδ), and Class IB (PI3Kγ). PI3Kα and β isoforms are ubiquitously expressed by almost all cell types, whereas PI3Kδ and γ are predominantly found in hematopoietic cells (Vanhaesebroeck et al., 2010). However, in the context of nociception at the DRG/spinal cord level, PI3Kγ isoform was found exclusively within DRGs in a subset of small- to medium-diameter isolectin IB4, TRPV1, and µ-opioid receptor-positive nociceptive neurons (Cunha et al., 2010; König et al., 2010; Leinders et al., 2014).
Duvelisib (DUV), also known as IPI-145 or Copiktra, is a novel small-molecule PI3Kδ/γ dual isoform-specific inhibitor. Because of the predominant expression of PI3Kδ and γ by hematopoietic cells, the major role of δ and γ isoforms is in the adaptive and innate immunity. Their inhibition has an immunosuppressive effect; therefore, DUV was originally developed as an oral treatment for various inflammatory diseases, autoimmune indications, and hematologic malignancies (Winkler et al., 2013; Boyle et al., 2014; Flinn et al., 2018). In September 2018, DUV was approved by the U.S. Food & Drug Administration (FDA, 2018b) for the treatment of relapsed or refractory chronic lymphocytic leukemia/small lymphocytic lymphoma (Blair, 2018).
Considering the predominant role of PI3Kδ and γ isoforms in inflammatory processes and PI3Kγ isoform expression in a subpopulation of nociceptive DRG neurons, we decided to test the hypothesis that PI3Kδ/γ isoform-specific inhibitor DUV will attenuate the painful PIPN development in an animal model.
Materials and Methods
Ethical approval
All procedures involving animals were approved by the local Institutional Animal Care and Use Committee and were consistent with the guidelines of the International Association for the Study of Pain, EU Directive 2010/63/EU for animal experiments and the National Institutes of Health Guide for the care and use of laboratory animals. All procedures were designed to minimize animal discomfort and to use the fewest number of animals needed for statistical analysis.
Animals
Adult male and female Wistar rats (8-10 weeks old, 250-350 g) were used for behavioral and pharmacology experiments. Only males were used for immunohistochemistry and intracellular calcium imaging in DRG neurons. Young male Wistar rats (6-7 weeks old, 150-200 g) were used for the behavioral, pharmacology, and patch-clamp electrophysiological experiments.
Adult male transgenic mice VGAT-ChR2-eYFP line 8, also known as B6.Cg-Tg(Slc32a1-COP4*H134R/EYFP)8Gfng/J (Purchased from The Jackson Laboratory; stock #014548) weighing ∼30 g were used for the behavioral, pharmacology, and patch-clamp electrophysiological experiments.
Animals were housed in plastic cages with soft bedding and maintained on 12 h light/12 h dark cycle at room temperature-controlled conditions. The experiments were conducted during the light phase of the cycle. Food and water were available ad libitum, except when under the food restrictions overnight before treatment via per oral (p.o.) gavage.
Animals were randomly distributed into three experimental groups: CTRL (Control group with vehicle pretreatment + vehicle treatment), PAC (vehicle pretreatment + PAC treatment) and DUV + PAC (DUV pretreatment + PAC treatment). The specific dosage of drugs and the number of animals used in each set of experiments are detailed in the relevant Materials and Methods/Results section.
PAC-induced painful peripheral neuropathy model and DUV treatment
PAC Mylan (Oncotec Pharma Produktion) was used to induce painful peripheral neuropathy in both rats and mice. An original clinically used stock solution of PAC (6 mg/ml) was diluted with 0.9% sterile saline to 2 mg/ml, just before injection. To replicate the original PAC formulation, a vehicle stock solution was made using 1:1 macrogolglycerol ricinoleate (Kolliphor EL, Sigma-Aldrich) and ethanol (Penta). Three successive doses of 8 mg/kg PAC were intraperitoneally administered in rats on days 0, 3, and 6, representing a final cumulative dose of 24 mg/kg. In mice, 2 mg/kg PAC (i.p.) was administered 4 times, every other day (on days 0, 2, 4, and 6), with the final cumulative dose 8 mg/kg. This dosage produces a preclinical model of chemotherapy-induced painful peripheral neuropathy in both species, as previously described (Liu et al., 2010; Li et al., 2015). In CTRL groups, animals were treated with a vehicle: the equivalent volume of Kolliphor EL and ethanol (1:1) in sterile saline.
DUV (LC Laboratories) stock solution (up to 200 mm) was prepared freshly in DMSO (Sigma-Aldrich) just before treatment. An appropriate dose of DUV was mixed with the vehicle (edible sunflower oil; 500 µl per rat, 100 µl per mouse) and administered via oral gavage into the stomach. Plastic feeding tubes (Instech Laboratories) 15 G × 78 mm and 20 G × 30 mm were used for rats and mice, respectively. DUV in doses 0.01 µg, 1 µg, 1 mg, and 10 mg/kg was administered as a pretreatment 30 min before the PAC unless otherwise stated. In some experiments, DUV (10 mg/kg) post-treatment 24 h after the PAC was also tested. In CTRL groups, animals were treated with the vehicle only.
Behavioral testing of mechanical withdrawal threshold and data analysis
Animals were placed on a stainless-steel wire mesh under clear acrylic glass cages in a quiet room and allowed to acclimate for ∼1 h. Paw withdrawal threshold (PWT) to tactile stimulation was tested manually using the electronic von Frey apparatus (IITC Life Sciences, Model 2390 Series). The probe tip of the electronic von Frey was applied 5 times to the plantar surface. The average value from each hind paw was calculated and then averaged in the experimental group. A quick flick, shaking or full paw withdrawal was considered as a response. Control PWT was tested in all groups on day −3 and 0 before any treatment. The behavioral testing was performed by a person blind to the treatment.
In all experiments, the dependent variable was the change in mean PWT, expressed as absolute change (in grams) or as percentage change from the control response on day 0, unless otherwise stated. To compare the effect of different treatments during the time on the development of mechanical hypersensitivity induced by PAC, a two-way repeated-measures ANOVA followed by Bonferroni post hoc test was used. Moreover, where males and females were compared, three-way ANOVA with sex (male, female), treatment (PAC, DUV + PAC), and days of treatment (−3, 0, 6, 8, 10, 14, 17, and 21) as factors and Bonferroni's post hoc test was used. GraphPad Prism 9.0.1 (GraphPad Software) was used to perform statistical analysis. Graphing was performed using GraphPad Prism and CorelDraw Graphics Suite 2020 software (Corel). p-value < 0.05 was considered statistically significant. All data are expressed as mean ± SEM. Detailed statistics are given in the figure legends.
Spinal cord slice preparation
The same experimental protocol for spinal cord slices preparation was used for adult mice and young rats, similar to our previous experiments (Spicarova et al., 2014b; Adamek et al., 2019). Laminectomy was performed under deep anesthesia with 3% isoflurane (Forane, Abbott), and the lumbar spinal cord was removed and immersed in oxygenated, ice-cold (∼4°C), dissection solution containing the following (in mm): 95 NaCl, 1.8 KCl, 7 MgSO4, 0.5 CaCl2, 1.2 KH2PO4, 26 NaHCO3, 25 d-glucose, and 50 sucrose. Animals were killed by subsequent medulla interruption and exsanguination. The spinal cord was fixed to a vibratome stage (VT 1200S, Leica Microsystems) using cyanoacrylate glue in a groove between two agar blocks. Acute transverse slices 300 μm thick were cut from L4-L5 segments and incubated in the dissection solution for 30 min at 35°C. Slices were then stored in a recording solution at room temperature (21°C-24°C) and allowed to recover for at least 1 h before the electrophysiological experiments. The recording solution contained the following (in mm): 127 NaCl, 1.8 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and 25 d-glucose. For the electrophysiological measurements, slices were transferred into a glass-bottomed recording chamber perfused continuously with the recording solution at room temperature at a rate of ∼2 ml/min. All extracellular solutions were saturated with carbogen (95% O2, 5% CO2) during the whole experiment.
Electrophysiology and optical stimulation
Whole-cell patch-clamp recordings were made from visually identified superficial dorsal horn neurons using a differential interference contrast microscope Zeiss Axio Examiner A.1 (Carl Zeiss Microscopy) equipped with infrared LED illumination and an infrared-sensitive camera Grasshopper 3 (Point Gray), connected to a standard personal computer. Patch pipettes were pulled from borosilicate glass tubing (Rückl Glass) on Pipette Puller P-97 (Sutter Instruments) and then filled with an intracellular solution for final resistance of 3.5-7.0 mΩ. The intracellular pipette solution contained the following (in mm): 125 gluconic acid lactone, 15 CsCl, 10 EGTA, 10 HEPES, 1 CaCl2, 2 Mg2ATP, and 0.5 NaGTP and was adjusted to pH 7.2 with CsOH. Voltage-clamp recordings in the whole-cell configuration were performed with an Axopatch 1D (Molecular Devices) amplifier and Digidata 1440A digitizer (Molecular Devices) at room temperature (∼23°C). Whole-cell recordings were low-pass filtered at 2 kHz and digitally sampled at 10 kHz. The series resistance of neurons was routinely compensated by 80% and was monitored during the whole experiment. Software package pCLAMP 11.0 (Molecular Devices) was used for data acquisition and offline analysis. Only postsynaptic currents with an amplitude of ≥5 pA (corresponding to at least twice the noise) were included in the frequency and amplitude analysis.
In experiments on rat spinal cord slices, AMPAR-mediated mEPSCs were recorded from visually identified superficial dorsal horn neurons in laminae I and outer II, clamped at −70 mV in the presence of 10 μm bicuculline methiodide, 5 μm strychnine (both from Sigma-Aldrich) and 0.5 μm TTX (Tocris Bioscience) in the bath solution. Recording of mEPSCs began ∼4 min after whole-cell access when the recorded current had reached a steady state. After recording of the control segment/basal activity (3 min), capsaicin (50 nm; Tocris Bioscience) was applied twice for 2 min with a 10 min interval in between the applications to study desensitization/tachyphylaxis of TRPV1-mediated capsaicin responses under different treatment conditions. Capsaicin was dissolved in DMSO (Sigma-Aldrich), which had a concentration of <0.1% in the final recording solution. Data segments of 2 min basal activity (before capsaicin) and 5 min after capsaicin application were evaluated. mEPSC occurrence was detected semiautomatically in segments of 10 s duration using the threshold search function in Clampfit 11.0 software and subsequently checked manually. The frequency of mEPSC is presented in absolute values (Hz) or is normalized as a percentage of the first capsaicin response (100%).
Spinal cord slices prepared from VGAT-ChR2-eYFP mice were used to study the excitatory and inhibitory inputs on excitatory dorsal horn neurons under different treatment conditions. The inhibitory/excitatory neurons were distinguished using 500-ms-long blue light (470 nm) photostimulation by M470F3 fiber-coupled LED with LEDD1B (Thorlabs) driver and triggered by Digidata 1440A digitizer (Molecular Devices). Only neurons lacking the ChR2-mediated plateau phase of the response induced by the 500 ms light pulse (putatively excitatory neurons) were used for further analysis. sEPSCs were recorded at −70 mV without any antagonist, and it was found that contamination by sIPSCs was negligible under our experimental conditions, which was further confirmed by the application of AMPAR antagonist CNQX (10 μm) and NMDA blocker AP5 (25 μm) at the end of sEPSC recording. GABAA receptor- and/or glycine receptor-mediated sIPSC and light-evoked (le-IPSC) were routinely recorded at 0 mV using the same intracellular and recording solution as sEPSC, in the presence of CNQX and AP5 during the whole experiment. le-IPSCs were recorded as a series of 10 5-ms-long photostimulations with a frequency of 0.1 Hz. These 10 responses were averaged for each cell/each experimental condition. Amplitudes, an area under the curve, rise time (10%-90%), and decay time (90%-10%) were analyzed. In some experiments, paired-pulse stimulation (1 ms photostimulation) with 50 ms interstimulus interval (ISI) was used to study short-term synaptic plasticity. A paired-pulse ratio (PPR) was calculated as a ratio between the amplitude of the second pulse (with subtraction of residual decay δ) and the first pulse (P2-δ/P1). In some experiments, GABAergic or glycinergic contribution to le-IPSC was studied using pharmacological blockade of glycine receptors (strychnine, 0.5 μm) or GABAA receptors (bicuculline methiodide 10 μm), respectively.
To evaluate statistically significant differences between groups, t test, one-way ANOVA (ordinary/or repeated-measures) followed by Tukey's post hoc test, or two-way repeated-measures ANOVA with Bonferroni's post hoc multiple comparison test was used, as specified in the relevant section. Graphing and statistical analysis were performed using GraphPad Prism 9.0.1 and CorelDraw Graphics Suite 2020 software. The criterion for statistical significance was p < 0.05, and all data are expressed as mean ± SEM. Detailed statistics are always given in the figure legends.
DRG cell culture preparation and calcium imaging
Adult Wistar rats were randomly distributed in three experimental groups: CTRL group (n = 4), PAC (n = 3), and DUV + PAC group (n = 3). Animals received the standard CTRL vehicle, PAC (3 × 8 mg/kg), or DUV (10 mg/kg) + PAC (3 × 8 mg/kg) treatment on days 0, 3, and 6, as described above. DRG cells were isolated from the animals on day 8 following the protocol of Malin et al. (2007) with appropriate modifications. Briefly, L3-L5 DRGs from both sides of the spinal cord of anesthetized adult male rats were excised and collected in ice-cold DMEM (Invitrogen, #10566-016). The intact DRGs were then enzymatically digested using a combination of 4 mg/ml (0.6% w/v) Collagenase Type II (Invitrogen, #17101015) and 3 mg/ml (∼2 U/ml) Dispase II (Sigma-Aldrich, #D4693) for 45 min in a 37°C incubator (oxygenated with 95% O2 and 5% CO2). After gentle washing of the partially digested DRG tissue in fresh DMEM, tissue was triturated and mechanically dispersed with Pasteur pipette and filtered using a 40 μm Corning cell strainer (Sigma-Aldrich, #CLS431750) to obtain DRG cell suspension. The DRG cells were pelleted by centrifugation at 200 × g, washed, and resuspended in growth media (DMEM/F-12 1:1, Invitrogen, #31331-028, 10% FBS, and 100 U/ml penicillin-streptomycin). DRG cells were then plated on 10 mm glass coverslips precoated with poly-D-lysine (1 mg/ml) and laminin (0.02 mg/ml) and incubated for 2 h in the 37°C for attachment. The unattached and/or dead cells were washed out by a fresh change of growth media after 2 h of plating, and the attached cells were stained and imaged the next day (18-24 h). All procedures were performed in sterile conditions in a culture hood.
Calcium imaging experiments were performed according to the established protocol (Svobodova et al., 2018) with appropriate modifications. Briefly, DRG cells were labeled with cell-permeant ratiometric Ca2+ indicator dye fura-2-acetoxymethyl ester (fura-2 AM; Invitrogen, # F-1221) in ACSF at a final concentration of 1 μm for 45 min in a 37°C incubator. The ACSF solution contained 142 mm NaCl, 3 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 10 mm HEPES, and 10 mm glucose adjusted to pH 7.4 with NaOH. After fura-2 loading, cells were washed 3× in ACSF, and fluorescence images were captured in real-time under a 20× water immersion objective using a Leica DM LB2 microscope (Leica Microsystems) during exposure to alternating 340 and 380 nm light excitation and emission at 510 nm (TILL Photonics). Image analysis was performed using MetaFluor software (Molecular Devices). The ratio of light intensity (340/380 nm) reflected changes in intracellular free Ca2+ concentration ([Ca2+]i) and was followed in ≥20 single DRG cells simultaneously at the rate of 1 Hz. During the experiment, cells were continually perfused with ACSF, and TRPV1 agonist (capsaicin, 200 nm) was applied focally for 3 s onto the cells under the FOV using a custom-made gravity-driven fast application perfusion system WAS-02 (Dittert et al., 2006). Recording of images was often paused in-between two capsaicin application to prevent exposure of cells to light and subsequent photobleaching.
Intracellular changes in Ca2+ with respect to two subsequent 200 nm capsaicin applications were measured 15 min apart in each DRG cell and were expressed as the change in the ratio of fluorescence intensity at 340/380 nm over time. For calculation of the results, the baseline ratio was averaged for 10 s before each capsaicin application for all cells considered for analysis. The amplitude of Ca2+ response to the first and second capsaicin application for an individual cell was derived by subtracting the base from the peak ratio after each capsaicin application, independent of one another. Cells that did not achieve their peak fluorescence within 5 s of capsaicin application were not included in the final analysis.
Immunohistochemical analysis of Akt kinase phosphorylation
Immunohistochemical analysis of Akt kinase phosphorylation was performed on DRG sections collected from adult male Wistar rats 1 h after acute treatment, or on days 7 and 21 of the PIPN model described above. Animals were randomly distributed in three experimental groups (CTRL, PAC, and DUV + PAC group) for each time point: 1 h acute treatment (n = 4, 4, 4), 7 d treatment (n = 3, 3, 3), and 21 d treatment (n = 3, 5, 3). CTRL animals received standard vehicle treatment. PAC-treated animals received vehicle gavage 30 min before PAC (8 mg/kg i.p.) as a single acute treatment or on days 0, 3, and 6. Finally, the DUV + PAC group was pretreated with DUV (10 mg/kg) 30 min before each PAC administration. Animals were deeply anesthetized at the appropriate time according to the different length of the treatment (1 h after the single treatment or on days 7 and 21) with a combination of ketamine (100 mg/kg, Narketan, Zentiva) and xylazine (25 mg/kg, Xylapan, Zentiva), perfused intracardially with saline followed by ice-cold 4% PFA. Both left and right L5 DRGs were removed and postfixed in 4% PFA at 4°C for 2 h, cryoprotected with 30% sucrose overnight, and cut in cryostat Leica CM3000 to 16-μm-thick slices. Every third DRG section was then processed for phosphorylated Akt (pAkt) immunohistochemistry. Briefly, sections were washed 3× for 10 min in PBS, blocked with 3% normal donkey serum (NDS) for 30 min at room temperature, and incubated overnight at 4°C with rabbit anti-pAkt (Ser473) (1:200; Cell Signaling Technology, #4060S) primary antibody in 1% NDS with 0.3% Triton X-100. After washing in 1% NDS (3× for 10 min), the sections were exposed to a donkey anti-rabbit Cy2-conjugated secondary antibody (1:400, Jackson ImmunoResearch Laboratories, #711-225-152) for 2 h. For visualization of the cell nucleus, incubation in bisbenzimide (Hoechst 33342, Sigma-Aldrich) for 3 min was used. Slices were mounted by DPX mounting medium. Pictures from all sections were captured using a digital camera on a fluorescence microscope (Olympus BX53) with UPlanApo (20×/0.70 NA) objective and analyzed offline using ImageJ software (National Institutes of Health) by an investigator blinded to the treatment. ROI containing only neuronal cell bodies (excluding nerve/root fibers) was outlined for each DRG section, and area of pAkt+ immunoreactivity (IR) was identified within the ROI using the Threshold function. To set the appropriate threshold value, we first obtained intensity values of multiple pAkt-IR+ cell bodies. At least, 5 sections were evaluated for each ganglion. Left and right DRGs were analyzed separately (n = 6-10 per experimental group; detailed statistics are given in the figure legend (Figs. 6, 7)). IR/ROI ratios were calculated and expressed as a percentage. To analyze the number of pAkt-IR DRG cells, all cell bodies in the DRG section were manually outlined, and pAkt-IR intensity and area of individual cell bodies were measured. Cell bodies were divided into pAkt-IR and pAkt-non IR groups based on the signal intensity. Only cells with visible nuclei were included in the analysis. Numbers of animals/DRGs/cells included in the analysis are as follows: CTRL 1 h (n = 4/8/4138), PAC 1 h (n = 4/8/4478), and DUV + PAC 1 h (n = 4/8/4754); CTRL 7 d (n = 3/6/2753), PAC 7 d (n = 3/6/3534), and DUV + PAC 7 d (n = 3/6/3189); CTRL 21 d (n = 3/6/2406), PAC 21 d (n = 5/10/4718), and DUV + PAC 21 d (n = 3/6/2806).
Immunohistochemical analysis of CD68-immunoreactive macrophages
Adult Wistar rats were randomly distributed in three experimental groups: CTRL group (n = 6), PAC (n = 8), DUV + PAC group (n = 9). Animals received the standard vehicle, PAC, or DUV + PAC treatment on day 0, 3 and 6, as described above. PAC was administered 3 × 8 mg/kg (i.p.) and DUV 10 mg/kg (p.o.) as 30 min pretreatment. Animals were perfused, and tissue was collected and processed (as described above) 24 h after the last treatment. Mouse anti-CD68 primary antibody [ED1] (1:100, Abcam, #ab31630) and a donkey anti-mouse AlexaFluor-488 secondary antibody (1:400, Jackson ImmunoResearch Laboratories, #715-545-151) were used. Slices were mounted by DPX mounting medium. Pictures were captured using a confocal microscope (Leica Microsystems, SP8). Multi-immersion objective HC PL APO (20×/0.75 NA), Ar multiline laser (488 nm; 65 mW), and HyD spectral detectors were used. Data were analyzed offline using ImageJ software (National Institutes of Health) by an investigator blinded to the treatment, as described above, using ROI containing only neuronal cell bodies area (excluding nerve/root fibers). CD68+ IR was analyzed in ROI using the Threshold function. Left and right L5 DRGs were analyzed separately (CTRL: n = 12, PAC group: n = 16, DUV + PAC group: n = 18).
Results
DUV treatment prevented PAC-induced mechanical hypersensitivity in both male and female Wistar rats
The first aim of the present study was to determine the effectiveness of DUV treatment in preventing mechanical hyperalgesia, a typical symptom of PIPN, in adult male Wistar rats. The model of PIPN was induced by three successive doses of PAC (8 mg/kg, i.p.) on days 0, 3, and 6 in combination with different doses of DUV (0.01 µg/kg to 10 mg/kg, p.o.; 30 min before or 24 h after treatment) (Fig. 1A). As shown in Figure 1B, von Frey measurement of PWT revealed significant mechanical hypersensitivity in PAC-treated animals compared with the CTRL group, whereas increasing doses of DUV significantly prevented PAC-induced hypersensitivity. After PAC treatment and pretreatment with low doses of DUV (0.01 and 1 µg/kg), PWT significantly decreased on days 6-21, compared with the CTRL animals (p < 0.05-0.001; for detailed statistics, see Fig. 1B legend). The administration of DUV 1 mg/kg and DUV 10 mg/kg pretreatment significantly prevented the effect of PAC. The highest dose of DUV (10 mg/kg) was also tested as 24 h after treatment in PAC-treated rats. This dose was still effective in preventing PIPN; nevertheless, the effect was not as strong as after the 30 min pretreatment with 10 mg/kg DUV. Therefore, the DUV 10 mg/kg administered as 30 min pretreatment was considered as the most effective in preventing the effect of PAC and used in the following experiment on rats.
DUV treatment prevented PAC-induced mechanical hypersensitivity in both male and female adult Wistar rats. A, Experimental design diagram. PIPN was induced by three successive doses of PAC (8 mg/kg, i.p.) on days 0, 3, and 6 as indicated by blue arrows. DUV was administered via oral gavage to the stomach in different concentrations (0.01 µg/kg to 10 mg/kg) as a 30 min pretreatment before PAC (orange arrows) or as a 10 mg/kg post-treatment 24 h after each PAC administration (dark red arrows). PWT to mechanical stimulation was measured from day −3 to day 21 as indicated by a pictogram of rat under the timeline. B, Males: PAC-treated animals developed significant mechanical hypersensitivity. Increasing doses of DUV significantly prevented PAC-induced decrease of PWT. Statistics: two-way repeated-measures ANOVA revealed a significant time × treatment interaction (F(42,462) = 13.91; p < 0.001). Bonferroni's post hoc multiple comparison test shows significant decrease of PWT after PAC treatment and after low doses of DUV (0.01 and 1 µg/kg) compared with CTRL group on days 8-21 (p < 0.05 to 0.001; n = 7, 13, 7, and 10 for CTRL, PAC, DUV 0.01 µg/kg, and DUV 1 µg/kg, respectively). DUV 1 mg/kg pretreatment (n = 11) and 10 mg/kg post-treatment (n = 15) were not different from the CTRL, but the effect was not as strong as the DUV 10 mg/kg pretreatment (n = 10), which was considered as the most effective in preventing the effect of PAC. C, Females: Animals received the same PAC treatment, and the most effective 10 mg/kg DUV pretreatment was tested. Whereas PAC produced significant mechanical hypersensitivity, DUV prevented its development. Statistics: two-way repeated-measures ANOVA revealed a significant time × treatment interaction (F(14,113) = 9.89; p < 0.001). Bonferroni's post hoc multiple comparison test shows significant differences between PAC and CTRL group on days 8, 10, 14, 17, and 21 (p < 0.001; n = 4, 11, and 7 for CTRL, PAC, and DUV + PAC, respectively). D, Comparison of PAC-induced hypersensitivity and relative effectiveness of DUV 10 mg/kg pretreatment in males and females. Statistics: three-way ANOVA with time (day of treatment), sex (male, female), and treatment (PAC, DUV + PAC) as factors revealed significant time × treatment × sex interaction (F(7,259) = 3.9; p < 0.001). In more detail, analysis revealed the main effect of treatment (F(1,37) = 127.9; p < 0.001) and time (F(7,259) = 96.0; p < 0.001), whereas the effect of sex was not statistically significant (F(1,37) = 0.72; p = 0.4). Moreover, any significant sex × time and sex × treatment interaction was not revealed. The only difference was found in time × treatment interaction (F(7,259) = 61.29; p < 0.001). These results suggest the treatment factor as the main source of variation and minor differences between males and females. It is in good agreement with Bonferroni's post hoc multiple comparison test, which revealed a significant effect of DUV in preventing PAC-induced changes in both males and females (p < 0.001; n = 10 and 7 for males and females). However, it did not reveal any significant changes between PAC-treated males and females (e.g., on days 17 and 21; n = 13 and 11 for males and females, respectively).
A substantial body of research shows significant sex-based differences in pain development, experience, and treatment (Paller et al., 2009; Mapplebeck et al., 2016). For this reason, the most effective 10 mg/kg DUV 30 min pretreatment was also tested in adult female Wistar rats. As shown in Figure 1C, PAC administration induced significant mechanical hypersensitivity on days 8, 10, 14, 17, and 21 (p < 0.001), whereas DUV pretreatment prevented its development. Comparison of PAC-induced hypersensitivity and relative effectiveness of DUV 10 mg/kg pretreatment in males and females (Fig. 1D) identified the treatment factor as the main source of variation, and no sex-based differences were found between the males and the females (three-way ANOVA, for detailed statistics, see Fig. 1D legend). These data indicate that administration of DUV before PAC effectively prevents the development of PIPN in both sexes.
DUV pretreatment in vivo (10 mg/kg) prevented PAC-induced sensitization of TRPV1 receptors to capsaicin in the rat SCDH
We have recently demonstrated that both in vitro and in vivo PAC treatment sensitized TRPV1-mediated capsaicin responses in the SCDH neurons and DRG neurons via the TLR4 (Toll-like receptor 4)/PI3K-dependent mechanism and that inhibition of PI3K signaling with nonspecific inhibitors wortmannin and LY-294002 prevented this PAC-induced sensitization of TRPV1 receptors (Li et al., 2015; Adamek et al., 2019).
Considering the PI3Kγ expression in TRPV1+ nociceptors (Cunha et al., 2010; König et al., 2010) and δ/γ-isoform specificity of DUV (Winkler et al., 2013), we tested the effect of the DUV (10 mg/kg; 30 min pretreatment) in the prevention of PAC-induced sensitization of TRPV1-mediated capsaicin responses. Six- to 7-week-old male rats received the PAC and DUV treatment as mentioned above and as shown in Figure 2A. Behavioral measurement of PWT confirmed the development of significant hypersensitivity in PAC-treated animals and the preventive effect of DUV (Fig. 2B). On day 8, spinal cord slices were prepared and used for the patch-clamp experiment. To test changes in sensitization and tachyphylaxis after repeated TRPV1 stimulation, capsaicin (50 nm) was applied twice for 2 min with a 10 min wash period between the first and the second application.
DUV pretreatment in vivo (10 mg/kg) prevented PAC-induced sensitization of TRPV1 receptors to repeated application of capsaicin in spinal cord slices. A, Experimental design schematic. PIPN was induced by intraperitoneal injection of PAC (3 × 8 mg/kg, i.p.). DUV (10 mg/kg) was administered as a pretreatment (30 min, p.o.) before the PAC injection. B, Behavioral measurement of mechanical PWT. PAC-treated animals developed a significant mechanical hypersensitivity, which was prevented by the DUV pretreatment. Statistics: two-way repeated-measures ANOVA revealed a significant time × treatment interaction (F(8,44) = 5.251, p < 0.001). Bonferroni's post hoc multiple comparison test shows a significant decrease of PWT after PAC treatment compared with the CTRL group on days 6 and 8 (p < 0.05 to 0.001). C-E, Representative recordings of mEPSCs recorded from SCDH neurons illustrate increased responses to repeated application of low concentrated TRPV1 agonist capsaicin (50 nm for 2 min; 10 min between first and second application) in the PAC-treated animals and the preventive effect of the DUV pretreatment. F, Detailed analysis of mEPSC frequency illustrates a significant increase after the PAC treatment, whereas the DUV pretreatment prevented this increase. Each line indicates averaged mEPSC frequency counted in 10 s intervals. Statistics: Two-way repeated-measures ANOVA revealed a significant time × treatment interaction (F(142, 2414) = 2.163, p < 0.001). Bonferroni's post hoc multiple comparison test revealed differences in specific time points between the PAC and CTRL groups (gray asterisks) as well as between the PAC and DUV + PAC groups (orange hash tag [#]; p < 0.05 to 0.001; n = 11, 15, and 11 for CTRL, PAC, and DUV + PAC group, respectively). G-I, Summary comparison of mEPSC frequencies during control activity (2-min-long interval) and after the first and the second capsaicin application (5-min-long intervals were compared). PAC treatment significantly increased control mEPSC frequency (G; F(2,34) = 3.5; p < 0.05). DUV pretreatment prevented increase of both the first (H; F(2,34) = 4.618, p < 0.05) and the second capsaicin response (I; F(2,34) = 11.94, p < 0.001) and prevented also PAC-induced reduction of the second capsaicin response tachyphylaxis, expressed as percentage of the second response relative to the first response (J; F(2,34) = 10.24, p < 0.001). Statistics: ordinary one-way ANOVA followed by Tukey's post hoc test. K-M, Analysis of mEPSC amplitudes did not show any significant changes, suggesting that mainly presynaptic mechanisms were involved (statistics: ordinary one-way ANOVA; for p value, see the graphs).
Representative mEPSC recordings from dorsal horn spinal cord neurons (Fig. 2C–E) and detailed mEPSC frequency plot (Fig. 2F) illustrate the PAC-induced increase/sensitization of the first capsaicin response and reduction of the second capsaicin tachyphylaxis, whereas DUV pretreatment prevented both of these effects. Detailed analysis revealed that PAC treatment significantly increased control/basal mEPSC frequency from 1.2 ± 0.2 Hz in the CTRL group to 3.4 ± 0.7 Hz in the PAC group (p < 0.05; Fig. 2G). In the DUV + PAC group (Fig. 2G), the PAC-induced increase of control mEPSC activity was decreased but not fully prevented by DUV (2.3 ± 0.5 Hz, p = 0.37). The mEPSC frequency of the first capsaicin response significantly increased from 11.7 ± 3.0 Hz in the CTRL group to 24.5 ± 4.1 Hz in the PAC group (p < 0.05). In neurons, recorded in the DUV + PAC group was the frequency significantly suppressed (11.5 ± 2.9 Hz, Fig. 2H; p < 0.05).
Further analysis showed that repeated application of capsaicin in the CTRL group leads to marked tachyphylaxis of the second capsaicin response, which reached frequency of 4.9 ± 0.8 Hz (Fig. 2I) and was only 48.1 ± 5.2% of the first response (Fig. 2J). In contrast, PAC treatment reduced the tachyphylaxis and increased the second capsaicin response frequency to 16.0 ± 2.6 Hz (p < 0.001), which was 70.7 ± 5.5% (p < 0.05) of the first response compared with the CTRL. The PAC-induced effect was completely prevented in the DUV + PAC group to 4.5 ± 1.0 Hz (Fig. 2I; p < 0.001) and 39.2 ± 4.3% (Fig. 2J; p < 0.001).
Examination of the mEPSC amplitudes did not show any significant changes in the control segment (Fig. 2K) either after the first (Fig. 2L) or the second capsaicin (Fig. 2M) application, suggesting that mainly presynaptic mechanisms were involved in the PAC-induced modulation of TRPV1 sensitivity to capsaicin as well as in the DUV treatment action.
PAC-induced sensitization of calcium responses to repeated application of capsaicin in lumbar DRG neurons was prevented by DUV pretreatment
To support our findings from the patch-clamp electrophysiology, an in vitro calcium imaging experiment in dissociated rat lumbar DRG neurons was performed to investigate the effect of PAC and DUV pretreatment at the DRG level. In vivo treatment paradigm was the same as in the patch-clamp experiment (Fig. 2A). Initial perfusion of capsaicin (200 nm; 3 s) on DRG neurons in the CTRL group resulted in a robust increase of intracellular calcium level [Ca2+]i, as evidenced by an increase of the 340/380 ratio (Fig. 3A,B; CTRL Caps 1), and displayed marked tachyphylaxis when reapplied 15 min later (Fig. 3A,B; CTRL Caps 2). PAC treatment significantly sensitized the first response as well as reduced the tachyphylaxis of the second response compared with the CTRL group. DUV in vivo pretreatment fully prevented these PAC-induced changes in sensitivity of DRG neurons to both the first and the second capsaicin application.
Calcium imaging experiments in dissociated DRG neurons confirmed the PAC-induced sensitization of TRPV1 receptors and the preventive effect of the DUV pretreatment in vivo. A, Representative calcium imaging traces of the 340/380 nm ratio after the first and the second capsaicin (200 nm) application in dissociated DRG neurons prepared from young male rats on day 8 after the treatment. The experimental paradigm was the same as in the patch-clamp experiment (see Fig. 2A). B, DRG neurons prepared from the PAC-treated animals compared with the CTRL and the DUV + PAC-treated animals revealed significantly increased capsaicin responses in both the first (F(2,390) = 5.556; p < 0.01) and the second response (F(2,410) = 24.13; p < 0.001). Statistics: Ordinary one-way ANOVA followed by Tukey's multiple comparison post hoc test (p < 0.05 to 0.001; n = 139, 119, and 135 for CTRL, PAC, and DUV + PAC group, respectively). C, PAC treatment significantly reduced tachyphylaxis of the second capsaicin response, whereas the DUV pretreatment significantly prevented these changes. Ordinary one-way ANOVA (F(2,390) = 27.45; p < 0.001) followed by Tukey's multiple comparison post hoc test (p < 0.001).
Finally, the effect of DUV in preventing PAC-induced reduction of tachyphylaxis is illustrated in Figure 3C where the second capsaicin response is expressed relatively as a percentage of the first one. In the CTRL group, the second capsaicin response reached 48.4 ± 1.6% with marked tachyphylaxis, whereas in the PAC group it significantly increased to 64.8 ± 1.7%. In the DUV + PAC group, DUV pretreatment showed a highly significant effect in reduction of the second response close to control level, 52.0 ± 1.6% (p < 0.001).
DUV prevented PAC-induced hypersensitivity and imbalance of the spontaneous inhibitory/excitatory neurotransmission in the SCDH in adult VGAT-ChR2-eYFP mice
Disturbance of the balance between excitatory and inhibitory mechanisms, for example, because of sensitization of the excitatory component or because of disinhibition can lead to pathologic pain states (Sivilotti and Woolf, 1994). An increasing body of evidence indicates that diminished inhibitory GABAergic and glycinergic neurotransmission at the SCDH level contributes significantly to the development of pain states (Sandkühler, 2009; Zeilhofer et al., 2012). The mechanisms of PIPN are extensively studied; nevertheless, little is known about how PAC treatment affects inhibitory synaptic control at the spinal cord level.
To study the effect of PAC on disinhibition in the SCDH (and the potential preventive effect of DUV), we next used a combination of electrophysiology and optical stimulation to detect/quantify changes in the excitatory/inhibitory synaptic transmission onto the laminae I/II neurons. We used male VGAT-ChR2-eYFP transgenic mice, which express an optimized channelrhodopsin (ChR-H134R fused with an eYFP fluorescent reporter protein) under the transcriptional control of the Slc32a1 gene promotor of vesicular inhibitory amino acid transporter (VGAT) specifically expressed in all inhibitory, GABAergic and glycinergic neurons (Zhao et al., 2011).
First, we performed behavioral experiments to elucidate the dose of DUV necessary to prevent the PIPN development in mice because different DUV pharmacokinetics have been reported in mice and rats (FDA, 2018a). PIPN was induced by four injections of PAC (2 mg/kg, i.p.) every other day. DUV was administered as a pretreatment (30 min, p.o.) before PAC in three different doses (1, 10, and 100 mg/kg) (Fig. 4A). Behavioral measurement of PWT (Fig. 4B) revealed the development of significant hypersensitivity in the PAC-treated group. While DUV pretreatment with concentrations 1 and 10 mg/kg showed no or very low efficacy in preventing PIPN, 100 mg/kg significantly blocked the development of hypersensitivity and did not differ significantly from the CTRL group (for detailed statistics, see Fig. 4B legend). Based on these findings, DUV 100 mg/kg pretreatment was considered as the most effective in preventing the effect of PAC in mice and only animals treated with 100 mg/kg DUV were used for spinal cord slices preparation followed by patch-clamp study.
DUV pretreatment in vivo (100 mg/kg) prevented PAC-induced mechanical hypersensitivity and disinhibition in the excitatory SCDH neurons in adult mice. A, Schematic of the treatment paradigm. PIPN was induced by intraperitoneal injection of PAC (2 mg/kg) every other day. DUV was administered in three different doses (1, 10, and 100 mg/kg) as a pretreatment (30 min, p.o.) before the PAC. Animals from the CTRL, PAC, and DUV (100 mg/kg) + PAC groups were subsequently processed on day 8 for the electrophysiological experiment. B, Behavioral measurement of mechanical PWT. PAC-treated animals developed a significant hypersensitivity. Increasing doses of DUV prevented the PAC-induced decrease of the PWT. Statistics: two-way repeated-measures ANOVA revealed a significant time × treatment interaction (F(20,110) = 7.07; p < 0.001). Bonferroni's multiple comparison post hoc test shows a significant decrease of PWT after PAC treatment and after low-dose DUV (1 and 10 mg/kg) compared with the CTRL group on days 4, 6, and 8 (p < 0.05 to 0.001). DUV 100 mg/kg was not different from the CTRL group and therefore considered as the most effective in preventing the effect of PAC. Only animals treated with DUV 100 mg/kg were used for the patch-clamp study. C, D, The inhibitory/excitatory neurons were distinguished by using a 500-ms-long blue light (470 nm) photostimulation. Only putative excitatory neurons without ChR2-mediated plateau phase of the response were used for the next analysis. E, Illustration of the sEPSC recorded at −70 mV without any antagonist in the recording solution. Contamination by sIPSC at −70 mV was negligible as confirmed by the application of CNQX (10 μm) and NMDA blocker AP5 (25 μm) at the end of sEPSC recording. F, G, Compared with the CTRL group, the PAC treatment significantly impairs the balance between the sEPSC and sIPSC frequencies as shown in the representative recordings and the graph (G; PAC group, paired t test; p < 0.001). H, sEPSC frequency was significantly increased in the PAC group (p < 0.01; n = 36) compared with both the CTRL (n = 28) and the DUV + PAC groups (n = 36; one-way ANOVA, F(2,97) = 7.8; p < 0.001). sIPSC frequency was significantly decreased after the PAC treatment compared with the CTRL (one-way ANOVA, F(2,97) = 3.9; p < 0.05); nevertheless, the preventing effect of the DUV treatment in the DUV + PAC group was not significant (p = 0.17). Frequencies plotted as an sEPSC/sIPSC ratio for each cell showed a significant shift of the ratio after the PAC treatment in favor of the sEPSC that was prevented by the DUV (one-way ANOVA, F(2,97) = 10.4; p < 0.001). Ordinary one-way ANOVA followed by Tukey's post hoc test was used for each comparison. I, Amplitude analysis (one-way ANOVA) of both sEPSC and sIPSC did not reveal any significant changes (one-way ANOVA, F(2,97) = 2.5; p = 0.085, for sEPSC and F(2,97) = 0.1; p = 0.9, for sIPSC).
Using photostimulation with 470 nm blue light, we were able to selectively, rapidly, and reversibly activate the population of inhibitory interneurons and distinguish between recording from inhibitory (ChR2+) and presumably excitatory neurons (ChR2–). Photostimulation for 500 ms induced in inhibitory neurons an action potential-independent photo-current with typical ChR2-mediated plateau phase (Fig. 4C), while in putatively excitatory neurons it induced an le-IPSC without ChR2 plateau (Fig. 4D), which was action potential-dependent, and therefore sensitive to TTX (data not shown). Only identified excitatory neurons were used for further recording to study the changes of inhibitory inputs, specifically in the lamina I/II of the SCDH.
sEPSCs were recorded at −70 mV without any antagonist in the recording solution. Contamination by sIPSCs at −70 mV was negligible under our experimental conditions as confirmed by application of AMPAR antagonist CNQX (10 μm) and NMDA blocker AP5 (25 μm) at the end of sEPSC recording (Fig. 4E). Subsequently, sIPSCs were recorded from the same neuron at 0 mV in the presence of CNQX and AP5 during the whole experiment.
Our data showed that PAC treatment significantly impairs the balance between spontaneous excitatory and inhibitory neurotransmission in the SCDH excitatory neurons, as illustrated by representative recordings (Fig. 4F) and summary graphs (Fig. 4G; PAC, p < 0.001).
Detailed analysis revealed that sEPSC frequency (Fig. 4H) significantly increased in the PAC group (3.4 ± 0.4 Hz, p < 0.01) compared with both CTRL (1.8 ± 0.3 Hz) and the DUV + PAC groups (1.7 ± 0.3 Hz). The sIPSC frequency (Fig. 4H) was after the PAC treatment significantly decreased (0.6 ± 0.1 Hz, p < 0.05) compared with the CTRL (1.4 ± 0.2 Hz). The DUV + PAC group was not significantly different compared with the CTRL (1.1 ± 0.3 Hz); nevertheless, the effect of DUV pretreatment shows only a partial and nonsignificant effect in the prevention of PAC-induced decrease of the sIPSC frequency (p = 0.17). Plotting the data as an sEPSC/sIPSC frequency ratio (Fig. 4H) for each cell well illustrates the imbalance between the excitatory and the inhibitory currents after the PAC treatment and a significant shift of the ratio in favor of the sEPSCs (p < 0.001). The DUV pretreatment significantly prevented this PAC-induced imbalance (p < 0.001).
Amplitude analysis of both sEPSC and sIPSC did not reveal any significant changes (Fig. 4I), which suggests that the discovered changes of sEPSC/sIPSC transmission were mediated primarily by presynaptic mechanisms.
DUV prevented PAC-induced disinhibition of glycinergic inputs in the population of excitatory SCDH neurons in VGAT-ChR2-eYFP mice
Following the sIPSC/sEPSC recording period, the total le-IPSC evoked by 5 ms, 470 nm photostimulation of the dorsal horn was recorded (Fig. 5A). As illustrated in Figure 5Aa-Ad, the PAC treatment significantly reduced the amplitude and area of le-IPSCs. In the CTRL group, le-IPSC had mean amplitudes of 0.72 ± 0.07 nA, whereas those in the PAC-treated group were significantly reduced to 0.5 ± 0.04 nA (p < 0.05). In recordings from the DUV + PAC group, the PAC-induced decrease was fully prevented and the mean le-IPSC amplitude was 0.77 ± 0.07 nA (Fig. 5Ac; p < 0.01). The same effect of PAC and DUV pretreatment was found when the le-IPSC area was analyzed (Fig. 5Ad). Average le-IPSC traces normalized to the control peak amplitude and superimposed (Fig. 5Ae) illustrate almost similar kinetics in all experimental groups. It is in good agreement with the results of rise time (Fig. 5Af) and decay time (Fig. 5Ag) analysis, which did not reveal any significant changes between the groups. Similar kinetics of the le-IPSCs suggest that the properties of postsynaptic receptors were unchanged following the PAC or DUV + PAC treatments and that presynaptic mechanisms may be responsible for the le-IPSC decrease, rather than changes in properties of postsynaptic GABAA or glycine receptors.
DUV pretreatment in vivo (100 mg/kg) prevented PAC-induced disinhibition of glycinergic neurotransmission in the excitatory SCDH neurons in adult mice. Aa, le-IPSCs evoked by 5 ms, 470 nm blue light stimulation of the dorsal horn. Individual gray traces represent an average of 10 le-IPSCs per individual cells. Thicker colored traces represent the average le-IPSC for each group: CTRL (n = 34), PAC (n = 48), and DUV (100 mg/kg) + PAC (n = 37). Ab, Overlapped le-IPSCs illustrate that PAC treatment significantly reduced both the le-IPSC amplitude (Ac; one-way ANOVA, F(2,116) = 6.9; p < 0.01) and the area (Ad; one-way ANOVA, F(2,116) = 5.5; p < 0.01) of the le-IPSC, whereas the DUV pretreatment in vivo prevented this PAC-induced decrease. Ae, Average le-IPSC traces (normalized to the control peak amplitude and superimposed) illustrate almost similar kinetics. It is consistent with the results of rise time (Af) and decay time (Ag) analysis, which did not reveal any significant changes between the experimental groups (one-way ANOVA, F(2,116) = 0.92; p = 0.39 for rise time, and F(2,116) = 0.86; p = 0.43 for decay time). Ba, Representative traces of paired-pulse responses; 1 ms stimulation and 50 ms ISI were used. Absolute amplitude of the first pulse was decreased after the PAC treatment (n = 15) compared with the control PPR (n = 24). This effect was prevented in the DUV + PAC group (n = 37), which is consistent with the le-IPSC (Ac). Traces with amplitudes normalized to the first pulse (Bb) and graph (Bc) represent a significant decrease of the PPR after the PAC treatment. This depression was probably caused by the depletion of inhibitory neurotransmitters after the PAC treatment. DUV pretreatment prevented this PAC-mediated effect (ordinary one-way ANOVA, F(2,73) = 10.0; p < 0.001). Ca, Representative traces illustrating the proportional contribution of glycinergic (GLY; green) and GABAergic (GABA; purple) currents to the total le-IPSC. Bicuculline (10 μm) was used for pharmacological isolation of the GLY component, whereas strychnine (0.5 μm) was used for isolation of the GABA component. Cb, Under the CTRL conditions, the proportion of GLY contribution to the total le-IPSC was significantly higher compared with the GABA (p < 0.001, unpaired t test, n = 8 and 9 for GLY and GABA, respectively). The GLY contribution was reduced after the PAC treatment, and the proportional contribution of GLY and GABA was almost balanced, without a significant difference (p = 0.21, unpaired t test, n = 13 for both GLY and GABA group). DUV prevented the PAC-induced decrease of the GLY contribution to the total le-IPSC (p < 0.001, unpaired t test; n = 20 and 10 for GLY and GABA group, respectively). Cc, Statistical comparison of the GLY component amplitudes of the total le-IPSC illustrates a significant reduction of the GLY amplitudes after the PAC (p < 0.05) and the protective effect of the DUV pretreatment (p < 0.001, one-way ANOVA, F(2,28) = 12,2; p < 0.001, followed by Tukey's post hoc test; n = 8, 13, and 20 for CTRL, PAC, and DUV + PAC, respectively). Cd, Statistical comparison of GABA component amplitudes did not reveal any significant changes (one-way ANOVA; n = 9, 13, and 7 for CTRL, PAC, and DUV + PAC, respectively). Da, Representative le-IPSC normalized to the peak amplitudes and superimposed illustrate the difference in the decay kinetics of the GABA and GLY components. Statistical analysis of the rise and decay time of both the GLY (Db) and GABA (Dc) component did not reveal any significant changes between the treatments (one-way ANOVA).
To study short-term synaptic plasticity and test the possible role of presynaptic changes in the decrease of the le-IPSCs, optical paired-pulse (PPR) stimulation protocol with 1 ms light stimulation and 50 ms ISI was used. Representative traces of PPR (Fig. 5Ba) and traces with amplitudes normalized to the first pulse (Fig. 5Bb) showed a significant decrease of the PPR to 0.48 ± 0.05 after PAC treatment (p < 0.001) compared with the control PPR 0.82 ± 0.04 (Fig. 5Bc). DUV pretreatment partially, but significantly, prevented this PAC-mediated effect (PPR 0.64 ± 0.05; p < 0.05; details in the figure legend, Fig. 5). These results suggest that the reduction of inhibitory synaptic transmission by PAC could be because of the depletion of neurotransmitter stores in the presynaptic endings of inhibitory interneurons.
To determine the role of GABAergic and/or glycinergic neurotransmission in this decrease, the proportional contribution of glycinergic and GABAergic currents to the total le-IPSC was further examined. In the following recordings, GABAA receptor blocker bicuculline (10 μm) was used for pharmacological isolation of glycinergic component, whereas glycine receptor antagonist strychnine (0.5 μm) was used for isolation of GABAergic component in a different subset of recordings. Representative traces (Fig. 5Ca) illustrate the different proportional contribution of glycinergic (GLY) and GABAergic (GABA) currents to the total le-IPSC under different experimental conditions. Under the CTRL conditions, the proportion of GLY contribution to the total le-IPSC was significantly higher (61.7 ± 7.0%) compared with GABA (26.2 ± 5.0%; p < 0.001; Fig. 5Cb). The GLY contribution was reduced after the PAC treatment, and the proportional contribution of GLY and GABA was almost balanced, without a significant difference (44.2 ± 5.3% vs 35.7 ± 3.9% for GLY and GABA, respectively; p = 0.21; Fig. 5Cb). DUV prevented the PAC-induced reduction of GLY contribution to the total le-IPSC (73.6 ± 3.1% vs 43.8 ± 6.2% for GLY and GABA group, respectively; p < 0.001; Fig. 5Cb). The comparison of GLY component amplitudes (Fig. 5Cc) illustrates a significant reduction of GLY amplitudes after PAC (0.19 ± 0.03 nA, p < 0.05) compared with CTRL (0.45 ± 0.08 nA) and the protective effect of DUV pretreatment (0.61 ± 0.07 nA, p < 0.001). The analysis of GABA component amplitudes (Fig. 5Cd) did not reveal any significant changes between the different groups (for detailed statistics, see the figure legend, Fig. 5).
The subsequent analysis was focused on the identification of possible changes in the GLY/GABA component kinetics. Representative le-IPSCs normalized to the peak amplitudes and superimposed (Fig. 5Da) illustrate the difference in the decay kinetics of the slow GABAergic and fast glycinergic component. Nevertheless, statistical analysis of rise time and decay time of both the GLY (Fig. 5Db) and the GABA components of le-IPSCs (Fig. 5Dc) did not reveal any significant changes between the different treatments.
These results demonstrate that one of the mechanisms contributing to the development of PAC-induced hypersensitivity may be PAC-induced glycinergic dysfunction in the dorsal horn inhibitory interneurons and subsequent imbalance between excitatory/inhibitory neurotransmission in excitatory dorsal horn neurons. Our results also showed that DUV pretreatment in vivo prevented these PAC-induced effects recorded in vitro.
DUV pretreatment prevented PAC-induced Akt phosphorylation
Based on the literature available, both DUV and PAC have only very limited blood–brain barrier (BBB) permeability (Yan et al., 2015b; FDA, 2018a); therefore, their primary effect is supposed to be mainly via peripheral mechanisms. It was reported that DRGs are less protected by the BBB, compared with the CNS (Allen and Kiernan, 1994) and therefore may be potentially more susceptible to the PAC and DUV effect. Phosphorylation of Akt/protein kinase is considered a reliable marker of PI3K signaling pathway activation (Zhuang et al., 2004; Pezet et al., 2008). Previously, we have shown that PAC activates PI3K signaling in DRG neurons and increases the level of pAkt following acute in vivo treatment (Adamek et al., 2019).
The following immunohistochemistry aimed to prove the effect of DUV in preventing PAC-induced pAkt phosphorylation in rat lumbar DRGs. Acute pAkt phosphorylation was determined 1 h after the PAC (8 mg/kg, i.p.) treatment. CTRL group received vehicle treatment and the DUV + PAC animals received DUV (10 mg/kg, p.o.) pretreatment 30 min before the PAC. Representative pictures of L5 DRG sections illustrate a significant increase of pAkt immunopositivity in the PAC group (Fig. 6Ba,Bb), compared with the vehicle-treated CTRL (Fig. 6Aa,Ab) and the DUV pretreated animals (Fig. 6Ca,Cb). Size distribution diagram in the PAC group (Fig. 6Bc) illustrates a significant increase of pAkt immunopositivity in the population of small diameter (15-25 µm) neurons compared with CTRL (Fig. 6Ac) and DUV + PAC group (Fig. 6Cc; for detailed statistics, see figure legend).
To determine the level of pAkt phosphorylation in time points relevant to the in vivo behavioral experiments, pAkt immunopositivity was also analyzed in rat lumbar DRG slices collected from animals on days 7 and 21. In these experiments, the treatment was the same as in the behavioral study (PAC group received three successive doses of 8 mg/kg PAC, i.p., on days 0, 3, and 6; DUV + PAC group received 10 mg/kg of DUV, p.o., 30 min pretreatment before the PAC and CTRL group received vehicle). Size distribution diagram in the PAC 7 d group (Fig. 6E) shows a significant increase of pAkt immunopositivity in the population of small-diameter neurons (20-25 µm) compared with CTRL 7 d (Fig. 6D) and DUV + PAC 7 d group (Fig. 6F). No significant increase of pAkt immunopositivity was observed in the size distribution diagrams of DRGs collected on day 21 after the initial treatment (Fig. 6G–I).
DUV (10 mg/kg) pretreatment prevented Akt phosphorylation in DRG neurons after PAC administration in vivo. Aa-Cc, The immunostaining was performed 1 h after the PAC (8 mg/kg, i.p.) treatment. Aa-Ac, Distribution of pAkt immunofluorescence (IR) in L5 DRG sections under the CTRL conditions (vehicle-treated animals). Size distribution diagram (Ac) of the pAkt+ and pAkt– neurons represents pAkt presence mainly in the population of small- and medium-diameter neurons. Ba-Bc, Compared with the CTRL situation, PAC treatment significantly increased the number of pAkt+ small-diameter neurons in the category of 15-20 µm cells (p < 0.001) and changed the positive/negative ratio in the category of 20-25 µm cells (p < 0.001 in CTRL, vs p = 0.9 in PAC). Ca-Cc, After the DUV p.o. pretreatment 30 min before the PAC i.p., the number of pAkt+ cells was significantly reduced. D-I, Distribution of pAkt IR in L5 DRG sections on days 7 (7D) and 21 (21D) after three successive doses of vehicle/PAC/DUV + PAC. D, Size distribution diagram of the pAkt+ and pAkt– neurons in CTRL group represents pAkt presence mainly in the population of small- and medium-diameter neurons. E, pAkt immunopositivity was significantly increased in small-diameter neurons in the category of 20-25 µm cells (p < 0.001) on day 7 in PAC group. F, DUV pretreatment reduced the number of pAkt+ small-diameter neurons on day 7. G-I, Size distribution diagrams of the pAkt+ and pAkt– neurons reveal similar distribution in between CTRL, PAC, and DUV + PAC groups on day 21 (2 weeks after the last treatment). Two-way repeated-measures ANOVA followed by Bonferroni's post hoc multiple comparison test was used for comparing the differences in the percentage of positive cells in each diameter category (p < 0.05 to 0.001). Scale bars: Aa, Ba, Ca, 200 µm; Ab, Bb, Cb, 40 µm.
The summary graph of the percentage of pAkt+ neurons (Fig. 7A) illustrates that, compared with the CTRL group (28.8 ± 0.8%), acute, 1 h PAC treatment dramatically increased the percentage of positive neurons within L5 DRGs (71.2 ± 0.8%; p < 0.001). In the DUV + PAC group, the number of positive neurons detected was significantly reduced and close to the control level (27.3 ± 0.8%). Similar results were obtained by the analysis of pAkt-immunopositive area (Fig. 7B). The pAkt-immunopositive area of DRG sections was significantly increased after the acute PAC (28.6 ± 2.0%; p < 0.05) compared with the CTRL (21.3 ± 1.4%), and reduced significantly with the DUV pretreatment (20.9 ± 1.6%).
DUV pretreatment prevented Akt phosphorylation in DRG neurons (the summary analysis of the data presented in the Fig. 6). A, Summary graph represents that the change in the percentage of the pAkt+ neurons induced 1 h after acute PAC administration was significantly reduced by the DUV pretreatment. Ordinary one-way ANOVA (F(2,21) = 421.5; p < 0.001) followed by Tukey's multiple comparison post hoc test (p < 0.001). B, The area of pAkt immunopositivity of the DRG sections was significantly increased after the 1 h PAC treatment and reduced with the DUV pretreatment. Ordinary one-way ANOVA (F(2,21) = 6.601; p < 0.01) followed by Tukey's multiple comparison post hoc test (p < 0.05). C, PAC (3 × 8 mg/kg, i.p.) induced increase in percentage of pAkt+ cells was observed also on day 7, and this effect was reduced by DUV pretreatment. Ordinary one-way ANOVA (F(2,15) = 33.61; p < 0.001) followed by Tukey's multiple comparison post hoc test (p < 0.05 to 0.001). D, pAkt+ area was increased after PAC treatment on day 7; this increase was prevented by pretreatment with DUV. Ordinary one-way ANOVA (F(2,15) = 36.22; p < 0.001) followed by Tukey's multiple comparison post hoc test (p < 0.001). E, F, No increase in number of pAkt+ neurons and pAkt+ area after PAC treatment was observed in DRG slices from animals on day 21. The only observed difference was a small decrease in the number of pAkt+ cells in the DUV + PAC 21 d group compared with PAC 21 d (p < 0.05). Ordinary one-way ANOVA (F(2,19) = 3.56; p = 0.048, for percentage of pAkt+ cells; F(2,19) = 1.83; p = 0.19, for percentage of pAkt+ area) followed by Tukey's multiple comparison post hoc test. For numbers of animals/DRGs/cells included in the analysis, see Materials and Methods.
In DRGs collected on day 7 (Fig. 7C), pAkt+ neurons represented 49.1 ± 1.9%, which is significantly more than 29.8 ± 1.5% observed in the CTRL 7 d group (p < 0.001). DUV pretreatment in the DUV + PAC 7 d group reduced the number of pAkt+ cells to 37.0 ± 1.6%. DRGs from the PAC 7 d group (Fig. 7d) also showed a robust increase in pAkt+ area (37.6 ± 1.1%), compared with the CTRL 7 d group (24.7 ± 1%; p < 0.001). This increase was significantly reduced in the DUV + PAC 7 d group (27.5 ± 1.3%).
Two weeks after the last treatment on day 21 (Fig. 7E), the only observed difference was a decrease in the number of pAkt+ cells in the DUV + PAC 21 d group (27.3 ± 1.9%) compared with the PAC 21 d. No significant changes in the pAkt+ area were observed on day 21.
These data imply that DUV effectively prevented PAC-induced increased activation of PI3K signaling in sensory DRG neurons, which can be one of the causes of sensory neurons sensitization and excessive excitatory neurotransmission at the spinal cord level. On day 21 (2 weeks after the last treatment), no significant effect of PAC on pAkt immunopositivity was observed.
DUV pretreatment prevented PAC-induced infiltration/proliferation of CD68-immunoreactive macrophages in the rat DRGs
Macrophages are fully differentiated cells of the mononuclear phagocytic lineage. Infiltration of monocytes from circulation and subsequent differentiation to macrophages in the DRGs as well as proliferation from resident macrophages is considered to play important role in the development and maintenance of neuropathic pain under different conditions (Ji et al., 2016; Yu et al., 2020), including PIPN (Huang et al., 2014; Zhang et al., 2016). In macrophages, PI3K signaling regulates the responses to different metabolic and inflammatory signals and modulates macrophage polarization (Sharif et al., 2019). In addition, PI3Kδ was reported to be involved in the regulation of the spreading and motility of macrophages (Mouchemore et al., 2013). Therefore, the next experiment aimed to test the hypothesis, that δ/γ-specific inhibitor DUV prevents the increased level of macrophages in the DRGs after PAC treatment.
We identified macrophages using an antibody against CD68, a lysosomal membrane protein that is expressed primarily in active phagocytic macrophages (Damoiseaux et al., 1994). We quantified CD68-immunopositive area in adult rat L5 DRG sections from CTRL (vehicle-treated), PAC (3 × 8 mg/kg, i.p.), and DUV + PAC-treated animals (30 min pretreatment with DUV, 10 mg/kg, p.o. before PAC). Animals were perfused, and tissue was collected 24 h after the last treatment (day 7).
The CD68 immunoreactivity in the PAC group (Fig. 8B) showed a significant increase compared with the CTRL (Fig. 8A), whereas the DUV pretreatment (Fig. 8C) significantly prevented PAC-induced increase of the CD68 immunopositivity. As summarized in the graph (Fig. 8D), the CD68+ area after the PAC (2.0 ± 0.1% of DRG) significantly increased by 43.6% compared with the CTRL group (1.4 ± 0.1% of DRG; p < 0.001) and formed aggregates around neuronal cell bodies. DUV pretreatment significantly prevented the PAC-induced increase of CD68 immunopositivity (1.5 ± 0.1% of DRG; p < 0.01).
DUV pretreatment prevented PAC-induced infiltration of CD68-immunoreactive macrophages into the DRG. Adult rats were perfused, and tissue was collected 24 h after the last treatment. PAC (3 × 8 mg/kg, i.p.) was administered on days 0, 3, and 6. DUV (10 mg/kg, p.o.) was administered as 30 min pretreatment. L5 DRG sections from the CTRL (A), PAC (B), and DUV + PAC (C) treated animals were examined immunohistochemically with a CD68 antibody against an intracellular lysosomal protein expressed in activated macrophages. D, Summary graph represents a significant increase in the CD68 immunoreactivity in the PAC group compared with the CTRL, whereas the DUV pretreatment significantly prevented PAC-induced macrophage infiltration into the DRG. Ordinary one-way ANOVA (F(2,43) = 9.729, p < 0.001) followed by Tukey's post hoc test (p < 0.01 to 0.001). Numbers of animals/DRGs included in the analysis: CTRL (n = 6/12), PAC (n = 8/16), and DUV + PAC (n = 9/18). Scale bars: A-C, 50 µm.
Discussion
PIPN accompanied with chronic neuropathic pain is a frequent unwanted side effect of patient treatment (Reyes-Gibby et al., 2009; Seretny et al., 2014). Recently, PI3K signaling was reported to be involved in the PIPN development (Adamek et al., 2019; Manjavachi et al., 2019; J. X. Huang et al., 2020).
This study is the first to reveal that orally available PI3Kδ/γ isoform-specific inhibitor DUV prevented the pain-like behavior and pro-nociceptive signaling in the PIPN model. DUV prevented the development of mechanical hyperalgesia in both males and females and PAC-induced imbalance of excitatory/inhibitory neurotransmission in the SCDH. We identified the role of TRPV1 in the PAC-induced excessive excitatory neurotransmission of DRG neurons and the reduction of inhibitory, mainly glycinergic, neurotransmission within the SCDH. DUV treatment prevented these adverse effects of PAC. These findings could provide a new strategy for the treatment of PIPN in patients, as DUV was approved for use in humans as Copiktra, albeit with a different diagnosis (FDA, 2018b).
Recent research brought strong evidence about sex differences in the development of neuropathic pain (Mogil, 2012). In our experiments, PAC produced similar PIPN in both sexes, which is in agreement with previous reports (Hwang et al., 2012; Brewer et al., 2020). We showed that DUV (10 mg/kg) pretreatment significantly reduced the development of mechanical hyperalgesia in male and female adult rats. A higher dose of DUV (100 mg/kg) was needed to prevent the development of mechanical hyperalgesia in male mice, probably because of the different pharmacokinetics. In mice, DUV is extensively metabolized by liver microsomes compared with rats. Also, the oral bioavailability of DUV is different and reaches 57% in rats and only 7% in mice (FDA, 2018a). As the very low dose of DUV (1 µg/kg) in rats also attenuated the mechanical hypersensitivity, we cannot exclude the possibility that, with higher dosing, also PI3Kα/β isoforms were affected.
DRGs are less protected by BBB than the brain and spinal cord and therefore are more vulnerable to chemotherapy-mediated neurotoxicity. That probably explains the predominance of peripheral mechanisms involved in PIPN (Cavaletti et al., 2000; Park et al., 2013). Based on the available literature, DUV was not detected in the brain 1 h after a single oral 5 mg/kg administration in rats, whereas it was widely distributed in peripheral tissues (FDA, 2018a). It suggests negligible BBB permeability and indicates that the primary effect of systemic DUV treatment could mainly affect peripheral mechanisms, including DRG neurons. The activation of DRG neurons by PAC was shown previously (Sisignano et al., 2016; Li et al., 2017), and TLR4 (Li et al., 2014; Yan et al., 2015b) and PI3K signaling-mediated modulation of TRPV1 receptors was reported (Adamek et al., 2019).
Immunohistological analysis of pAkt in lumbar DRGs showed that DUV prevented PAC-induced activation of PI3K signaling in small- to medium-sized DRG neurons 1 h after acute PAC administration, as well as on day 7 after three consecutive doses of PAC/DUV. This effect is in good agreement with the results of our patch-clamp and calcium imaging experiments and can explain the sensitization/tachyphylaxis of TRPV1-mediated responses to capsaicin. PI3K signaling was shown to produce strong sensitization of TRPV1-mediated responses (Zhang et al., 2005; Zhu and Oxford, 2007). A similar mechanism of PI3K-mediated sensitization of TRPV1 to capsaicin was recently reported by our group after both in vitro and in vivo PAC treatment (Adamek et al., 2019). PI3K catalytic subunit p85 may directly bind to the TRPV1 ankyrin repeat domain. Increased TRPV1 trafficking to the plasmatic membrane of cell bodies and presynaptic endings may explain increased responsiveness to capsaicin (Stein et al., 2006; Stratiievska et al., 2018). Similar PI3K-dependent regulation was found in AMPARs (Wigerblad et al., 2017), acid-sensing ion channel 1 (Duan et al., 2012), and several types of voltage-gated Ca2+ channels (Viard et al., 2004). Increased sensitivity of presynaptic TRPV1 receptors demonstrated in this study is also likely to contribute to behavioral changes, as TRPV1 antagonist was shown to prevent the PAC-induced mechanical hypersensitivity (Li et al., 2015).
Moreover, we showed that DUV prevented PAC-induced infiltration of CD68+ macrophages into the DRGs. In DRG neurons, PAC increases the expression of monocyte chemoattractant protein 1 (MCP-1/CCL2) and fractalkine (CX3CL1) responsible for the macrophage infiltration into DRGs (Huang et al., 2014; Zhang et al., 2016). Activated macrophages may modulate the excitability of DRG neurons by proinflammatory/pronociceptive cytokines and chemokines as TNFα and IL-1β (Spicarova et al., 2011; Ji et al., 2016) and contribute to TRPV1 sensitization/upregulation (Spicarova et al., 2014a).
The PI3K/Akt signaling is essential in macrophage reprogramming and M1/M2 phenotype switching (Malyshev and Malyshev, 2015). It should be emphasized that both PAC and DUV inhibit M2-like and induce a switch to M1-like, tumoricidal, proinflammatory, and rather pronociceptive phenotype (Horwitz et al., 2018; Wanderley et al., 2018). However, DUV was reported to inhibit colony-stimulating factor 1 (CSF1)/PI3K signaling in monocytes/macrophages (FDA, 2018c). Macrophage chemoattractant CSF1 is expressed de novo by DRG neurons during neuropathic pain (Guan et al., 2016). CSF1 signaling significantly affects the process of infiltration of circulating monocytes/peripheral macrophages to the SCDH and DRGs, which critically contributes to neuropathic pain development (Peng et al., 2016). We presume that the decrease of CD68+ immunoreactivity and antinociceptive effect of DUV reported here was mediated by inhibiting macrophage infiltration from circulation, rather than by switching between M1/M2 phenotype of resident DRG macrophages.
Disinhibition within the SCDH critically contributes to neuropathic pain development (Zeilhofer, 2008). The role of disinhibition in the PIPN remains unclear, and mainly the GABAergic component was studied. Activation of spinal microglia after systemic PAC treatment impaired GABAergic transmission via IL-1β and TLR4 signaling (Yan et al., 2015a, 2019). PAC also diminished GABA-induced hyperpolarization in SCDH via Na+-K+-2Cl– cotransporter-1 (NKCC1) upregulation and intracellular Cl– level impairment (Chen et al., 2014). PAC also increased GABA reuptake from the synaptic cleft via GABA transporter GAT-1 upregulation (Masocha and Parvathy, 2016). Interestingly, transplant-mediated GABAergic tone enhancement prevented PAC-induced mechanical and heat hyperalgesia (Braz et al., 2015).
This is the first study reporting PAC-induced impairment of glycinergic synaptic transmission within the SCDH. Together with sensitization of TRPV1+ nociceptors, this may result in imbalanced excitatory/inhibitory neurotransmission on excitatory SCDH neurons.
Our data suggested that PAC induces disinhibition by a presynaptic mechanism, probably via depletion of neurotransmitters in the presynaptic endings of inhibitory interneurons. Subsequent analysis of GABAergic/glycinergic contribution to the total le-IPSC revealed a significant reduction of the glycinergic le-IPSC component following the PAC treatment. Reduction of glycinergic control in the SCDH via presynaptic mechanism was also shown in models of peripheral inflammation (Muller et al., 2003) and nerve injury (Imlach et al., 2016). Glycinergic feedforward inhibitory circuits in the laminae II-III were implicated as a “gate control” mechanism for pain, and its impairment leads to mechanical allodynia (Lu et al., 2013). The glycinergic transmission plays an essential role in the SCDH inhibitory transmission, and its contribution to evoked inhibitory currents was 60%-70% under normal conditions (Foster et al., 2015; Imlach et al., 2016), which is in good agreement with our findings where the glycinergic current was ∼62% of the le-IPSC. Following the PAC treatment, this glycinergic contribution was significantly reduced to ∼44%. However, DUV pretreatment in vivo prevented this PAC-induced decrease (∼74%).
Considering the limited BBB permeability of DUV (FDA, 2018a), we hypothesize that DUV prevents changes in spinal cord inhibitory circuits indirectly, by preventing PAC-induced sensitization of primary afferent nociceptors. Decreased invasion/activation of CD68+ macrophages following DUV treatment may also be a contributing factor. The excessive C-fiber activity critically contributes to the development of central sensitization (Latremoliere and Woolf, 2009). Following PAC treatment, the release of many pronociceptive neuromodulators (e.g., CCL2, CX3CL1, substance P, and BDNF) increased in the spinal cord, which contribute to the development and maintenance of disinhibition, central sensitization, and pain behavior (Peters et al., 2007; Zhang et al., 2012, 2013; Huang et al., 2014; Spicarova et al., 2014b; Chiba et al., 2016). Several mechanisms for loss of inhibitory neurotransmission in neuropathic pain have been reported: upregulation of microglial P2X4 purinoceptor and overproduction of BDNF resulting in downregulation of K+-Cl– cotransporter (Coull et al., 2005; Wu et al., 2019), substance P, and NK1 receptor signaling, leading to spinal endocannabinoids release and glycine and GABA release inhibition via CB1 receptor activation (Jennings et al., 2001; Drew et al., 2009; Pernia-Andrade et al., 2009). Preventing these changes at the spinal cord level as a consequence of the DUV peripheral effect may explain its antinociceptive action.
Although DUV/Copiktra was approved for clinical use, side effects should be mentioned. Because of PI3Kδ/γ involvement in the immune system, DUV may cause serious infections, diarrhea, colitis, skin reactions, and neutropenia (Verastem, 2018).
In conclusion, this study showed, for the first time, that PI3Kδ/γ-specific inhibitor DUV prevented PAC-induced pronociceptive signaling in the DRG and SCDH and pain-like behavior in two different species. This work also revealed, for the first time, PAC-induced weakening of inhibitory, mainly glycinergic, control within the SCDH neurons. Our data suggest that the pathologic pain after the PAC administration is at least partially mediated by sensitization of DRG neurons, possibly because of modulation of TRPV1 and also because of invasion of circulating macrophages. Enhanced excitatory nociceptive drive together with decrease of spinal inhibitory circuits leads to the imbalance of the excitatory/inhibitory neurotransmission. DUV prevents these changes, presumably by blocking the PI3K signaling in DRG neurons and macrophages, thus inhibiting the PIPN development. Considering the approval of DUV for use in humans, we suggest a possible new strategy for preventing PIPN in patients with the preemptive use of DUV. Repurposing of a drug is a coveted goal for the drug-development industry; therefore, there may be a significant translational potential of our findings.
Footnotes
This work was supported by the Grant Agency of the Czech Republic GACR 20-19136S and RVO67985823. We thank Katerina Kramerova for technical support and help with performing behavioral and immunohistochemical experiments.
The authors declare no competing financial interests.
- Correspondence should be addressed to Jiri Palecek at jiri.palecek{at}fgu.cas.cz
