Introduction

Neuropathic pain is a debilitating chronic pain condition that results from injury or disease of the somatosensory system. Since chronic pain is refractory to currently available treatments, the development of effective treatments is a major clinical challenge ^1,2. Accumulating evidence from studies using models of neuropathic pain indicates that peripheral nerve injury causes various inflammatory responses in the nervous system that critically contribute to the development of neuropathic pain ^3,4. Especially, pro-inflammatory cytokines [e.g., interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNFα)] produced by various non-neuronal cells such as macrophages at injured nerves and dorsal root ganglion (DRG), and glial cells (mainly microglia and astrocytes) in the spinal dorsal horn (SDH) modulate function of primary afferent sensory neurons and SDH neurons, leading to an increase in the excitability of pain signaling neural pathway ^3–6. Along with these findings, much attention has been paid to the pain-relieving role of anti-inflammatory cytokines ⁷. These include IL-10, the role of which in chronic pain has been extensively studied ⁷. Another anti-inflammatory cytokine with pain-relieving effects in diverse models, including neuropathic pain, is IL-4. Intraplantar or systemic administration of IL-4 has been shown to attenuate pain behavioral responses to bradykinin ⁸, carrageenin ⁸, TNFα ⁸, zymosan ⁹ and acetic acid ⁹ in normal animals. In models of neuropathic pain, single or repeated administration of IL-4 around the injured sciatic nerve reduced behavioral pain hypersensitivity ^10–12. These effects of IL-4 have been proposed to involve a decrease in the production of inflammatory mediators ¹¹, polarization of macrophages toward an anti-inflammatory phenotype at injured nerves ^10,11, increases in the production and release of IL-10 ¹¹ and opioid peptides ^10,12 from macrophages, and inhibition of the increased action potentials of DRG neurons ¹³. In addition to the diverse actions of IL-4 at the peripheral level, the central nervous system, including the spinal cord, is recognized as an important locus of action of IL-4 in pain control. Intrathecal administration of IL-4 prevents the development of mechanical pain hypersensitivity in several neuropathic pain models ^13,14. The target cells for the action of IL-4 intrathecally administered could be microglia since, within the SDH of nerve-injured rats, IL-4 receptor α chain (IL-4Rα) is predominantly expressed in microglia ¹⁴. In line with this, intrathecal IL-4 activates signal transducer and activator of transcription 6 (STAT6; an intracellular signal transduction molecule downstream of IL- 4Rα) in SDH microglia ¹⁴. Intrathecal IL-4 or other manipulation to increase spinal IL-4 during early phase after nerve injury suppresses cellular indexes for reactive microglia ¹⁴, IL-1β and Prostaglandin E2 release, and microglial p38 phosphorylation ¹⁵, all of which are required to develop pain hypersensitivity ⁴. In addition to the effect of IL-4 on pain development, it is noteworthy that intrathecal administration of IL-4 during the late phase (e.g., 2 weeks or later after nerve injury) leads to recovery from a behavioral pain-hypersensitive state that develops following nerve injury ¹⁴. Despite the remarkable therapeutic potential of spinal IL-4 for ameliorating established neuropathic pain, little is known about its mechanism of action. Given that the reactive states of microglia occur early after nerve injury (e.g., cell number and expression of proinflammatory genes) and subside in the late phase ^4,16,17, it is possible that the pain-resolving effect of IL-4 involves a mechanism other than the previously reported suppression of inflammatory responses in activated microglia.

In this study, using two different models of neuropathic pain, we demonstrated that intrathecally administered IL-4 during the late phase changes microglia to a CD11c⁺ state, which has recently been shown to be necessary for the spontaneous remission of behavioral hypersensitivity associated with neuropathic pain ¹⁸, and, importantly, that this is required for the pain-relieving effect of intrathecal IL-4. Consequently, our findings uncover a mechanism for the ameliorating effect of spinal IL-4 on already-developed neuropathic pain and provide evidence at a preclinical level that IL-4 could induce the emergence of CD11c⁺ microglia with a function that resolves neuropathic pain.

Results

Intrathecal treatment of IL-4 increases CD11c⁺ microglia in the SDH and alleviates pain hypersensitivity after SpNT

To examine the effect of IL-4 on the behavioral remission of neuropathic pain, we used mice with spinal nerve transection [SpNT; a model of neuropathic pain ^19,20] and repeated the administration of IL-4 to their intrathecal spaces from day 14 post-SpNT, when the paw withdrawal threshold (PWT) decreased (indicating that mechanical pain hypersensitivity had been developed) (Fig. 1A). Intrathecal treatment with IL-4 increased the PWT in the hindpaw ipsilateral (but not contralateral) to the SpNT (Fig. 1A). Notably, this behavioral remission of neuropathic mechanical hypersensitivity persisted for at least 5 days after the last intrathecal injection of IL-4. Because we previously showed that CD11c⁺ microglia emerging in the SDH after SpNT are necessary for the spontaneous remission of pain behavior that gradually occurs around 3 weeks after nerve injury ¹⁸, we explored the mechanism of action of spinal IL-4 with a focus on CD11c⁺ microglia in the SDH. In Itgax-Venus mice, in which CD11c⁺ cells are visualized using the fluorescent protein Venus ^18,21, SpNT increased the number of Venus⁺ (CD11c⁺) cells in the SDH ipsilateral to the injury (Fig. 1B). Consistent with our previous data ¹⁸, almost all CD11c⁺ cells were immunohistochemically positive for ionized calcium-binding adapter molecule 1 (IBA1) and the purinergic P2Y12 receptor (P2Y12R) (Fig. 1B and C), which are markers for myeloid cells and microglia, respectively ^22,23. We found that intrathecal administration of IL-4 to SpNT mice enhanced the increase in CD11c⁺ cells in the SDH (Fig. 1B-D). These cells also expressed both IBA1 and P2Y12R (Fig. 1B and C), indicating that CD11c⁺ cells increased by intrathecal IL-4 were microglia. Quantitative flow cytometry analysis revealed a significant increase in CD11c⁺ microglia in the SDH of IL-4-treated SpNT mice (Fig. 1D). In contrast, IL-4 did not significantly change the total number of microglia in the SDH (Fig. 1D). These findings suggest that CD11c⁺ microglia may be involved in the mechanism by which intrathecal treatment with IL-4 attenuates pain hypersensitivity.

Figure 1.

IL-4-induced remission of pain hypersensitivity requires spinal CD11c⁺ microglia

To determine whether CD11c⁺ microglia are required for pain remission induced by IL-4, we used Itgax-DTR-EGFP mice in which the diphtheria toxin (DTX) receptor (DTR) and enhanced green fluorescence protein (EGFP) are expressed on CD11c⁺ cells ^18,24, and administered DTX intrathecally to these mice to deplete CD11c⁺ microglia in the spinal cord. Intrathecal IL-4 also induced behavioral remission of mechanical hypersensitivity in Itgax-DTR-EGFP mice, but the pain-reducing effect was eliminated by intrathecal injection of DTX on day 17 post-SpNT (Fig. 2A). The depletion of EGFP⁺ (CD11c⁺) microglia in the SDH by DTX injection was confirmed (Fig. 2B), indicating the necessity of spinal CD11c⁺ microglia for the pain-relieving effect of intrathecal IL-4. IL-4 also alleviated pain hypersensitivity in female mice, which was also dependent on CD11c⁺ microglia (Fig.3). In addition to SDH, CD11c⁺ cells have been shown to be present at the site of injury and in the DRG ¹⁸. Thus, we examined the involvement of CD11c⁺ cells in the DRG in the pain-relieving effect of intrathecal IL-4 using our previously reported strategy that enables the selective depletion of CD11c⁺ cells in the periphery ¹⁸. The intraperitoneal administration of DTX at a low dose (2 ng/g mouse) in SpNT mice treated with IL-4 on day 17 efficiently eliminated CD11c⁺ cells (also positive for IBA1) in the DRG (Fig. 4A). However, the number of CD11c⁺ microglia in the SDH were not significantly different between the PBS- and DTX-treated groups (Fig. 4B). Under these conditions, we found that the intraperitoneal administration of DTX had no effect on pain hypersensitivity suppressed by intrathecal IL-4 (Fig. 4C). These data indicate that intrathecally administered IL-4 induces microglia to be in a CD11c⁺ state, and that this induction is necessary for the ameliorating effect of IL-4 on neuropathic pain.

Figure 2.

Figure 3.

Figure 4.

CD11c⁺ microglia appearance in the SDH is blunted in SNI model

To extend the therapeutic potential of CD11c⁺ microglia induced by IL-4 in neuropathic pain, we employed another model of neuropathic pain [speared nerve injury (SNI); transection of tibial and common peroneal nerves] ²⁵. Consistent with previous data ²⁵, SNI induced a long-lasting pain-hypersensitive state without spontaneous remission, at least until the last day of testing (day 56 post-SNI) (Fig. 5A), which was in stark contrast to the time course of pain hypersensitivity in the SpNT model. We found that the number of CD11c⁺ microglia in the SDH at 14 days after nerve injury was lower in the SNI model than in the SpNT model (Fig. 5B). Quantitative analysis by flow cytometry confirmed that there were fewer CD11c⁺ SDH microglia in the SNI mice (Fig. 5C). A blunted appearance was also observed on days 35 and 56 post-SNI. In contrast, the total number of microglia in the SDH was comparable between the two models at all time points tested, suggesting an impairment of the nerve injury-induced appearance of CD11c⁺ microglia in the SNI model. Furthermore, insulin-like growth factor 1 (IGF1) has been identified as a factor that is highly expressed in CD11c⁺ microglia and is necessary for their pain-remitting effect ¹⁸. We quantified Igf1 mRNA expression in the spinal tissue or fluorescence-activated cell sorting (FACS)-isolated SDH microglia from nerve-injured mice (Fig. 6). While Igf1 expression in sorted microglia increased in both models after injury, its levels were lower in microglia from SNI mice than in those from SpNT mice (Fig. 6D). In addition, an increase in Igf1 mRNA expression in the tissue homogenate of the upper half of the SDH, where CD11c⁺ microglia accumulate ¹⁸, was not observed in tissues from the SNI model (Fig. 6E). Moreover, in FACS-isolated CD11c^high SDH microglia that highly expressed IGF1, the expression of Igf1 mRNA was significantly lower in the SNI model (Fig. 6F). Thus, given that either the depletion of CD11c⁺ microglia or knockout of their IGF1 prevents the spontaneous remission of pain in the SpNT model ¹⁸, the long-lasting pain-hypersensitive state in the SNI model could be related to the blunted increase in CD11c⁺ microglia in the SDH and their lower expression of IGF1.

Figure 5.

Figure 6.

IL-4 alleviates SNI-induced pain hypersensitivity in CD11c⁺ microglia-and IGF1-dependent manners

Based on the above findings, we tested whether IL-4 could increase the number of CD11c⁺ microglia and resolve pain hypersensitivity in the SNI model. Intrathecal treatment with IL-4 in SNI mice from day 14 to day 16 significantly increased the number of CD11c⁺ microglia on day 21, almost all of which were positive for IBA1 and P2Y12R (Fig. 7A-C). Similar to the data obtained for SpNT mice, the total number of microglia in the SDH were not significantly different between PBS- and IL-4-treated SNI mice (Fig. 7C). Behaviorally, intrathecal IL-4 treatment alleviated the SNI-induced mechanical hypersensitivity (Fig. 7D). Furthermore, the depletion of CD11c⁺ microglia in the SDH of IL-4-treated SNI mice by intrathecal administration of DTX on day 17 negated the ameliorating effect of IL-4 on pain hypersensitivity (Fig. 8A and B). Moreover, the IL-4’s effect was not observed in Cx3cr1^CreERT2/+;Igf1^flox/flox mice treated with tamoxifen (Fig. 8C), a treatment that enables selective knockout of the Igf1 gene in tissue-resident Cx3cr1⁺ cells ²⁶, the majority of which are microglia in the SDH ¹⁸. Overall, intrathecally administered IL-4 exhibited a pain-remitting effect in the SNI model, which was dependent on CD11c⁺ microglia and IGF1.

Figure 7.

Figure 8.

Discussion

In this study, we demonstrated for the first time that CD11c⁺ microglia are an essential target of IL-4 administered intrathecally to induce the behavioral remission of pain hypersensitivity caused by peripheral nerve injury. The pharmacological effect of intrathecal IL-4 appears to be mediated by its direct action on spinal microglia, as the expression of IL-4Rα in the SDH has been shown to be predominant in microglia after nerve injury ¹⁴. This is also supported by the data that activation of STAT6 (the downstream molecule of IL-4Rα) after intrathecal IL-4 selectively occurs in spinal microglia ¹⁴ and that primary cultured microglia treated with IL-4 become CD11c⁺ and express IGF1 ^27–29. Furthermore, based on our data showing that IL-4 increased the number of CD11c⁺ microglia without affecting the total number of microglia, it is conceivable that IL-4 acts on microglia and changes their state to CD11c⁺ microglia (rather than the proliferation of CD11c⁺ microglia themselves), leading to behavioral remission of neuropathic pain.

In addition to SDH, the peripheral effects of IL-4 on neuropathic pain have been reported in several pain models. IL-4 applied to injured peripheral nerves, such as sciatic nerves, ameliorates neuropathic mechanical hypersensitivity ¹¹. In sciatic nerves, IL-4Rα expression is upregulated in macrophages that accumulate in injured nerves ¹¹. Although intrathecal administration of IL-4 may affect cells in the DRG, our findings obtained from mice with depletion of CD11c⁺ IBA1⁺ cells (presumably macrophages) in the DRG (but not SDH) suggest that the cells required for intrathecal IL-4 to ameliorate pain hypersensitivity are CD11c⁺ microglia in the SDH, but not peripheral CD11c⁺ macrophages. Furthermore, perineurally administered IL-4 can act on macrophages, which leads to a change in their state from pro-to anti-inflammatory via a STAT6- dependent mechanism ¹¹ and further to a release of opioid peptides that are key factors in the suppression of neuropathic pain ¹⁰. In the SDH, IGF1 appears to be necessary for the pain-relieving effect of CD11c⁺ microglia induced by IL-4. This is supported by data obtained from microglia-selective IGF1-knockout mice (Cx3cr1^CreERT2/+;Igf1^flox/flox mice treated with tamoxifen). Although Cx3cr1⁺ cells, other than CD11c⁺ microglia, also lack Igf1 in these mice, given Igf1 expression is much higher in CD11c⁺ microglia than in CD11c^neg microglia ¹⁸, IGF1 from CD11c⁺ microglia would play an important role in IL-4- induced pain remission. Thus, while IL-4 acts on macrophages and microglia in the DRG/injured nerves and the SDH, respectively, the molecular basis of its ameliorating effects on neuropathic pain hypersensitivity is different.

Another notable finding of this study was that the emergence of CD11c⁺ spinal microglia following nerve injury was impaired in the SNI model, which did not exhibit spontaneous remission of behavioral pain hypersensitivity. This raises the possibility that the reduced ability of spinal microglia to shift toward the CD11c⁺ state in the SNI model contributes, at least in part, to long-lasting pain hypersensitivity because SpNT mice with depleted CD11c⁺ spinal microglia have been shown to exhibit persistent pain hypersensitivity ¹⁸. Considering our finding that the total number of spinal microglia was comparable between the SpNT and SNI models, the signal(s) for microglia to transition to the CD11c⁺ state, rather than to the initial activation after nerve injury, could be different in these models. The mechanisms underlying this difference require further investigation, but we highlight that even under such conditions, IL-4 administered intrathecally induces the appearance of CD11c⁺ microglia in the SDH and exerts a pain-remitting effect in a manner that dependent on CD11c⁺ microglia. These data suggest that spinal microglia in the SNI model retain the ability to respond to IL-4, allowing microglia to be in a CD11c⁺ state that remits neuropathic pain and broadly extends their therapeutic potential to neuropathic pain.

This study demonstrated that intrathecal IL-4 administration in SpNT mice increased the number of CD11c⁺ microglia in the SDH, which is essential for the pain-relieving effect of intrathecal IL-4. Furthermore, in another neuropathic pain model (SNI), which exhibits a long-lasting neuropathic pain state, the nerve injury-induced CD11c⁺ microglia appearance was blunted, but intrathecal IL-4 increased their appearance and ameliorated behavioral pain hypersensitivity in a CD11c⁺ microglia-dependent manner. Our study revealed the mechanism by which intrathecal IL-4 ameliorates pain hypersensitivity after nerve injury and provides evidence for a strategy to promote the emergence of CD11c⁺ microglia for treating neuropathic pain.

Materials and Methods

Mice

C57BL/6 mice were purchased from Charles River Japan (Kanagawa, Japan). Itgax-Venus mice (B6.Cg-Tg(Itgax-Venus)Mnz/J) ²¹, Itgax-DTR-EGFP mice (B6.FVB- 1700016L21Rik^{Tg(Itgax-DTR/EGFP)57Lan/J}) ²⁴, Cx3cr1^CreERT2/+ mice (B6.129P2(Cg)-Cx3cr1^{tm2.1(cre/ERT2)Litt}/WganJ) ²⁶, Igf1^flox/flox mice (B6.129(FVB)-Igf1^tm1Dlr/J) ³⁰ were purchased from Jackson Laboratory (Bar Harbor, ME). The data were obtained from male mice except for the data shown in Figures 1D, 3, and 7C. For induction of Cre recombinase, Cx3cr1^CreERT2/+; Igf1^flox/flox mice were injected subcutaneously with 4 mg of tamoxifen (Sigma, St. Louis, MO) dissolved in 200 μL corn oil (Wako, Osaka, Japan) twice with a 48-hr interval. Four weeks later when gene-deleting CX3CR1⁺ cells in peripheral tissues including the DRG has been shown to be replaced by non-deleting cells ^26,31, the mice were subjected to nerve injury. All mice used were aged 5–14 weeks at the start of each experiment, and were housed individually or in groups at a temperature of 22±1°C with a 12-hr light–dark cycle, and were fed food and water ad libitum. All animal experiments were conducted according to relevant national and international guidelines contained in the ‘Act on Welfare and Management of Animals’ (Ministry of Environment of Japan) and ‘Regulation of Laboratory Animals’ (Kyushu University) and under the protocols approved by the Institutional Animal Care and Use committee review panels at Kyushu University.

Peripheral nerve injury

We used two injury models, spinal nerve transection (SpNT) ^18,32, which is a modified spinal nerve injury model ¹⁹, and spared nerve injury (SNI) ²⁵. For the SpNT model, under isoflurane (2%) anesthesia, a small incision at L3–S1 was made. The paraspinal muscle and fat were removed from the L5 traverse process, which exposed the parallel-lying L3 and L4 spinal nerves. The L4 nerve was then carefully isolated and cut while leaving the L3 spinal nerve intact. The wound and the surrounding skin were sutured with 5-0 silk. For the SNI model, according to a previously reported method ³³, under isoflurane (2%) anesthesia, the skin on the left thigh was open, and terminal branches of the sciatic nerve were exposed by separating the thigh muscle layer using scissors and blunt forceps. Two of the three branches, the tibial and common peroneal nerves, were tightly ligated with 7-0 Nylon (NM-01, WASHIESU MEDICAL) and a small portion of the nerves distal to the ligation were removed while leaving the sural nerve intact. The wound and the surrounding skin were sutured with 5-0 silk.

Immunohistochemistry

According to our previously reported method ¹⁸, mice were deeply anesthetized by i.p. injection of pentobarbital and perfused transcardially with phosphate buffered saline (PBS) followed by ice-cold 4% paraformaldehyde/PBS. Transverse L4 spinal cord sections (30 μm) or L4 dorsal root ganglion (DRG) sections (20 μm) were incubated for 48 hrs at 4 °C with primary antibody for IBA1 (1:2000; Cat# 234 308, Synaptic Systems), P2Y12R (1:2000; Cat# AS-55043A, AnaSpec), or GFP (1:1000; Cat# 598, MBL Life science). Tissue sections were incubated with secondary antibodies conjugated to Alexa Fluor 405 (1:1000, Abcam), 488, or 546 (1:1000, Molecular Probes) and mounted with Vectashield. Three to five sections from one tissue were randomly selected and analyzed using an LSM700 Imaging System (Carl Zeiss, Japan).

Behavioral test

To assess mechanical sensitivity, calibrated von Frey filaments (0.02– 2.0 g, North Coast Medical, USA) were applied to the plantar surfaces of the hindpaws of mice with or without PNI ¹⁸ and the 50% PWT was determined using the up–down method ³⁴.

Intrathecal injection

Under 2% isoflurane anesthesia, a 30G needle attached to a 25- μL Hamilton syringe was inserted into the intervertebral space between L5 and L6 spinal vertebrae in mice, as previously described ^18,35. Recombinant Murine IL-4 was purchased from Peprotech (Cat# 214-14). IL-4 (40 ng/μL in PBS, 5 μL/mouse) or PBS as a control were intrathecally injected once a day for 3 days from day 14 post-PNI. DTX was purchased from Wako (Cat# 048-34371) and was injected intrathecally (0.1 ng/μL in PBS, 5 μL/mouse) or intraperitoneally (2 ng/g mouse) into Itgax-DTR-EGFP mice. Only mice whose hypersensitivity was attenuated by IL-4 treatment were used for the DTX- induced cell depletion experiment shown in Figure 4C (10 out of 12 mice were included) and Figure 8B (12 out of 15 mice were included).

Flow cytometry

As previously described ¹⁸, mice were deeply anesthetized by i.p. injection of pentobarbital and perfused transcardially with PBS to remove the circulating blood from the vasculature. The spinal cord was rapidly and carefully removed from the vertebral column and placed into ice-cold Hanks’ balanced salt solution (HBSS). The 3– 4 lumbar (L3/4) segments (2 mm long) of the spinal dorsal horn (SDH) ipsilateral and contralateral to nerve injury were separated. Unilateral spinal tissue pieces were treated with pre-warmed 0.8-mL enzymatic solution [0.2 U/mL collagenase D (Cat#11088866001; Roche) and 4.3 U/mL dispase (Cat# 17105041; GIBCO)] in HBSS containing 2% fetal bovine serum (FBS) for 30 min at 37 °C. The tissues were homogenized by passing through a 23G needle attached with a 1-mL syringe and were further incubated for 15 min at 37 °C. After that, the tissues were homogenized by passing twice through a 26G needle, and the enzymatic reaction was stopped by adding EDTA (0.5 M). To count the number of CD11c⁺ (Venus⁺ or EGFP⁺) microglia, cell suspension was blocked by incubating with Fc Block (Cat# 553142; BD Biosciences) for 5 min at 4 °C and immunostained with CD11b-AlexaFluor 647 (M1/70; 1:1000; Cat#557686, BD Biosciences) and CD45-PE (1:1000; Cat# 103106, Biolegend) for 30 min at 4 °C in the dark. After washing, the pellet was resuspended in ice-cold HBSS-FBS and filtered through a 35-μm nylon cell strainer (BD Biosciences) to isolate tissue debris from the cell suspension. The total number of microglia (CD11b⁺ CD45⁺ cells) in the L3/4 spinal cord or SDH was analyzed using CytoFLEX SRT (Beckman Coulter) and FlowJo software (TreeStar). Microglia with Venus fluorescence higher in intensity than that observed in microglia of wild-type mice were analyzed as CD11c⁺ microglia.

Fluorescent activated cell sorting (FACS)

Spinal tissues were removed and homogenized as described above. Myelin debris was removed from the cell suspension using Myelin Removal Beads II and a MACS LS column (Miltenyi Biotec, Bergisch-Gladbach, Germany) according to the manufacturer’s protocol and our previously reported method ¹⁸. After centrifugation (300 × g, 10 min, 4 °C), the cells were resuspended in HBSS containing 10% FBS. The cell suspension was blocked by incubating with Fc Block and immunostained with CD11b-Brilliant Violet 785 (1:1000; Cat# 101243, BioLegend), CD206-APC (1:200; Cat# 141708, BioLegend), and CD45-PE (1:1000; Cat# 103105, BioLegend) for 30 min at 4 °C in the dark. After washing, cell suspension was treated with 7-aminoactinomycin D (7-AAD; Miltenyi Biotec) and incubate for 10 min on ice for viability staining prior to cell sorting. CD11b⁺ CD45⁺ CD206^neg 7-AAD^neg-singlet cells are gated as live microglia and sorted using FACSAria III (BD Biosciences) or CytoFLEX SRT. The sorted cells were directly collected in lysis buffer for total RNA extraction using the Quick-RNA Micro-Prep kit (ZYMO). CD11c^high microglia were collected as cells with Venus fluorescence higher than the median Venus fluorescence of all CD11c⁺ microglia ¹⁸

mRNA extraction from spinal cord homogenates

Mice were deeply anesthetized by pentobarbital and perfused transcardially with ice-cold PBS followed by RNAlater™ Stabilization Solution (Cat# AM7021, Invitrogen). The isolated SDH tissue around the boundary between L3 and L4 segments was isolated and separated into the ipsilateral and contralateral sides, and the upper half portion of the SDH of each side was dissected as described previously ¹⁸. Total RNA was extracted from tissue homogenates using Trisure (CAT# BIO-38032, Bioline, USA) according to manufacturer’s protocol and purified with the Quick-RNA Micro-Prep kit (ZYMO).

qPCR

As described previously ¹⁸, the extracted RNA from the sorted cells or the tissue was transferred to reverse transcriptional reaction with Prime Script reverse transcriptase (2680B, Takara, Japan). Quantitative PCR (qPCR) was performed with Premix Ex Taq™ (Cat# RR390B, Takara, Japan) using QuantStudio® 3 (Applied Biosystems, USA). Expression levels were normalized to the values for Actb. The sequences of TaqMan primer pairs and probe are described below: Actb: 5’-FAM-CCTGGCCTCACTGTCCACCTTCCA-TAMRA-3’ (probe), 5’- CCTGAGCGCAAGTACTCTGTGT-3’ (forward primer), 5’-CTGCTTGCTGATCCACATCTG-3’ (reverse primer); Igf1: 5’- /56- FAM/TCCGGAAGC/ZEN/AACACTCACATCCACAA/3IABkFQ/-3’ (probe), 5’- AGTACATCTCCAGTCTCCTCA-3’ (forward primer), 5’- ATGCTCTTCAGTTCGTGTGT-3’ (reverse primer).

Quantification and statistical analysis

All data are shown as mean ± s.e.m. Statistical significance was determined using the unpaired t-test, two-way ANOVA with post hoc Tukey’s multiple comparison test or one-way ANOVA with post hoc Tukey’s multiple comparison test using GraphPad Prism 7 software. Differences were considered significant at P<0.05.

Acknowledgements

We would like to thank Editage for editing a draft of this manuscript. This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grants JP19H05658 (M.T.), JP20H05900 (M.T.) and JP24H00067 (M.T.), by the Core Research for Evolutional Science and Technology (CREST) program from AMED under Grant Number 23gm1510013h (M.T.) by Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP24ama121031 (M.T.). T.M. was supported by AMED (JP20gm6310016, JP21wm0425001, JP23gm1910004, JP23jf0126004), JSPS (KAKENHI JP21H02752, JP22H05062), The Mitsubishi Foundation, Daiichi Sankyo Foundation of Life Science, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for Research on Metabolic disorders, Ono Pharmaceutical Foundation for Oncology, Immunology and Neurology, The Nakajima Foundation, The Uehara Memorial Foundation and Takeda Science Foundation. K.K. was supported by JSPS (KAKENHI JP23K19403, JP24K18621), JST (ACT-X Grant Number JPMJAX232B), Chugai Foundation for Innovative Drug Discovery Science, and The Ichiro Kanehara Foundation.

Additional information

Author Contributions

K.K. designed experiments, performed almost all experiments, analyzed the data, and wrote the manuscript. R.S. performed some experiments and analyzed the data. K.H. assisted some experiments. T.M. provided advice for some experiments. M.T. conceived this project, designed experiments, supervised the overall project, and wrote the manuscript. All of the authors read and discussed the manuscript.