Microglia and macrophages contribute to the development and maintenance of sciatica in lumbar disc herniation : PAIN

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Microglia and macrophages contribute to the development and maintenance of sciatica in lumbar disc herniation

Lu, Xuana; Chen, Lunhaoa; Jiang, Chaoa; Cao, Keleib,c,d; Gao, Zhihuab,c,d; Wang, Yuea,*

Author Information
PAIN 164(2):p 362-374, February 2023. | DOI: 10.1097/j.pain.0000000000002708

1. Introduction

Lumbar disc herniation (LDH) often leads to back pain and sciatica.12 Symptomatic LDH occurred in approximately 1% to 3% of the general populations,11,29 adding a heavy burden to the health care system.9 Characterized by hyperalgesia and allodynia, sciatica is the focus in LDH-related clinical practice. Current conservative treatments often lack either satisfactory efficacy or predictable prognosis.38,65 Surgery may be indicated for those failed conservative treatments, yet residual leg pain remained in one-third of patients in the long term.13 Pathomechanisms underlying LDH-induced sciatica, therefore, deserve further investigations.

Mechanical compression of nerve roots by the herniated discs may be the main cause of sciatica.33,49 Yet, nerve root compression is often inconsistent with clinical symptoms.45,47 Moreover, the size of the herniated disc and the degree of compression are weakly associated with pain score.17 Increasing evidence demonstrated that inflammation, induced by the herniated nucleus pulposus (NP) tissues, may be indispensable in the pathogenesis of sciatica. Before the occurrence of disc herniation, the NP tissues have undergone a long-term degeneration process.52 In LDH patients, the broken annulus fibrosus (AF) leads to the exposure of the avascular and immune-privileged NP tissues to the immune system, triggering immune cell infiltration, inflammatory cytokines secretion, and neovascularization.35,41,52 These inflammatory responses activate neuron–immune interactions in both the peripheral nervous system (PNS) and the central nervous system (CNS) and may increase nociceptive sensitivity through primary afferents and spinal cord neurons, contributing to pain.8,68 Yet, the mechanism leading to sciatica largely remains inexplicit.

Macrophages and microglia play important roles in host–pathogen defense, tissue repair, and immune regulation.18,63 Evidence suggested that macrophages in the dorsal root ganglia (DRG) and microglia in CNS are important to the initiation and maintenance of neuropathic pain after peripheral nerve injury.14,16,66 Whether and how macrophages and microglia are engaged in LDH-induced sciatica remains largely unknown.

Appropriate animal models are crucial to unveil the pathogenesis of various diseases. Several rat LDH models have been established by compressing the nerve root with autologous NP tissues31,44 or stainless steel.26 However, transgenic tools and genetic manipulations are limited in rats, conferring a disadvantage in uncovering the mechanistic clues of the disease. Moreover, only nondegenerated NP tissues were used in all the rat LDH models to compress the nerve root.43,46 Yet, it is well-recognized that LDH occurs after disc degeneration, accompanied by a progressive structural failure of the intervertebral disc with substantial involvements of inflammation.1 Models using nondegenerated NP tissues may not be able to fully simulate the pathological processes and characteristics in LDH patients. Rather, models using degenerated NP tissues would be more appropriate for LDH studies.

In this study, we established a mouse model of LDH using degenerated disc to compress the nerve roots. The current study aimed to determine the changes of macrophages and microglia in LDH-induced sciatica and further to characterize the initiation and maintenance of sciatica after LDH. In addition, transcriptional changes in DRG were analyzed with RNA sequencing (RNA-seq) to identify candidate targets for LDH-induced sciatica.

2. Materials and methods

2.1. Animals

Adult C57BL/6J male and female mice (8-12 weeks old; Shanghai SLAC Laboratory Animal Corporation, China) were used in this study. All mice were housed under a 12-hour light/dark cycle with controlled room temperature (23 ± 1°C) and unlimited access to food and water. All surgical and experimental procedures were approved by the Tab of Animal Experimental Ethical Inspection at the author's institute. Animal surgeries were performed according to the Guidelines of the International Association for the Study of Pain.69

2.2. Antibodies and reagents

The following primary antibodies were used: rat anti-F4/80 (Cat# 14-4801-82; Invitrogen, Carlsbad, CA), rabbit anti-Iba1 (Cat# 019-19741; Wako Chemicals, Japan), goat anti-Iba1 (Cat# NB100-1028; Novus Biologicals, Littleton, CO), mouse anti-ATF3 (Cat# sc-81189; Santa Cruz, CA), rabbit anti-CD68 (Cat# ab125212; Abcam, CAmbridge, United Kingdom), goat anti-CSF1 (Cat# AF416; R&D systems, Minneapolis, MN), and rabbit anti-CSF-1R (Cat# sc-692; Santa Cruz).

The following secondary antibodies were used: Alexa Fluor 488-donkey anti-goat (Cat# A11055; Invitrogen, Carlsbad, CA), Alexa Fluor 488-donkey anti-rabbit (Cat# A21206; Invitrogen), Alexa Fluor 488-donkey anti-mouse (Cat# A21202; Invitrogen), Alexa Fluor 555-donkey anti-mouse (Cat# A31570; Invitrogen), Alexa Fluor 555-donkey anti-rabbit (Cat# A31572; Invitrogen), and Cy3-donkey anti-rat (Cat# 712-165-153; Jackson ImmunoResearch, West Grove, PA).

2.3. A needle puncture–induced disc degeneration model in mice

Before the experiments, mice were habituated to the housing facility for 7 days. Needle puncture of the lumbar disc was conducted as previously described,35 and the mice were fasted for 12 hours before surgery. Mice were anesthetized with sodium pentobarbital (100 mg/kg) intraperitoneally and placed on a warming pad under aseptic conditions. Mice were placed in a supine position, and the abdomen was shaved and sterilized. Under a stereoscopic microscope (SZX7; Olympus), a 1- to 1.5-cm midline longitudinal incision was made. The gut, connective tissues, vessels, and psoas major muscles were gently retracted to expose the ventral aspect of the L4/L5 intervertebral disc. A 27-gauge needle, with a stopper at the depth of 1 mm, was used to puncture the disc. After the needle tip was punctured into the disc center, the needle was remained there for 1 minute. The incision was then closed with 5-0 silk sutures. Four weeks after the surgery, the punctured disc (L4/L5, degenerated disc) and cranial intact disc (L3/L4, nondegenerated disc) were harvested. The AF were removed and the central portion of NP was cut into histological pieces (∼1 mm3), which were further used in mouse models of LDH for nerve root compression.

2.4. Mouse models of lumbar disc herniation

Mice were anesthetized with sodium pentobarbital (100 mg/kg) and then placed in a prone position with the dorsal skins shaved and sterilized. The line connecting bilateral crista iliaca was used as a reference to identify the spinous processes of L4 and L5 vertebrae. A dorsal midline incision was made to expose the spinous processes and the left multifidus muscles was detached to expose the left laminae of L4 and L5. Under a stereoscopic microscope, approximately half of the left L4 lamina and L4/L5 facet joint were removed to expose the left L4 nerve roots and DRG. A piece of NP tissues, nondegenerated or degenerated, was filled into the foramen to compress the left existing L4 nerve root and DRG to make a conventional LDH model and a modified LDH model, respectively. The sham group underwent the same surgical exposure of the nerve root and DRG without NP implantation.

2.5. Histological evaluation

Under anesthesia, mice were perfused with saline and subsequently with 4% paraformaldehyde. The lumbar spine, spinal cord, and DRG were collected and postfixed in 4% paraformaldehyde for 8 hours. The lumbar spine was decalcified in 10% ethylenediaminetetraacetic acid for 14 days. All collected tissues were dehydrated in 30% sucrose in phosphate buffer saline (pH = 7.2) for 7 days. Samples were embedded in optimal cutting temperature compound. Sagittal or transverse sections in 15 μm thickness were acquired using a freezing microtome (NX50; Thermo Fisher Scientific, Waltham, MA).

Midsagittal or transverse sections of the lumbar spine were stained with hematoxylin and eosin (H&E) and safranin O-fast green using standard protocols. Under a microscope (BX61; Olympus), the histological images of AF, NP, nerve root, and DRG were acquired. Histological evaluation of disc degeneration was performed using Rutges' approach, which measured disc height, morphologies of endplate and AF, the boundary between AF and NP, and the cellularity and matrix of NP in H&E and safranin O-fast green sections.54

2.6. Immunofluorescence analysis

Immunofluorescence staining was performed as previously reported.40 Shortly, sections were antigen-retrieved in citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) at 95°C for 20 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes at room temperature optionally. After blocking with 10% (wt/vol) BSA, sections were incubated overnight at 4°C with the following primary antibodies: rat anti-F4/80 (1:1000), rabbit anti-Iba1 (1:800), goat anti-Iba1 (1:800), mouse anti-ATF3 (1:200), rabbit anti-CD68 (1:500), goat anti-CSF1 (1:500), and rabbit anti-CSF-1R (1:600). Sections were washed with 0.5% Tween-TBS and incubated with secondary antibodies for 2 hours at room temperature. 4′,6-Diamidino-2-phenylindole (C1005; Beyotime, China) was used to label cell nuclei. Images were acquired using a confocal microscope (FV1200; Olympus). For each mouse, 3 sections (with an interval of 5 sections) were evaluated (ImageJ; National Institutes of Health) to measure the density of macrophages and microglia in the L4 DRG and lumbar spinal dorsal horn (SDH). Three observational fields (10,000 μm2) were randomly selected in DRG and SDH (laminae I-II), and cells were counted by a coauthor who was blinded to the group assignment. F4/80 in L4 DRG or Iba1 in SDH counterstained with 4′,6-diamidino-2-phenylindole was counted as a single cell. Measurements were averaged for quantitative measurements of macrophages and microglia, respectively.

2.7. PLX5622 administration

To ablate microglia and macrophages, mice were fed with PLX5622 (colony-stimulating factor 1 receptor (CSF1R) antagonist) formulated diet (1200 ppm; Plexxikon, Inc, Berkeley, CA) 7 days before the LDH modeling surgery and sustained till 0, 7, 14, and 28 days after surgery. The control diet (AIN-76A; Research Diets) was supplied when PLX5622-formulated diet was stopped. Control mice were fed with control diet.

2.8. RNA sequencing analysis

To acquire adequate amounts of DRG tissues for RNA-seq analysis, degenerated NP tissues was placed next to bilateral L4 and L5 nerve roots. The transcriptomic analysis included 5 mice each in the sham and LDH groups. In each mouse, the bilateral L4 and L5 DRG were pooled to provide a single independent sample. RNA was isolated using RNeasy micro kit (74004; QIAGEN), and DNA was removed using DNase I digestion, following the manufacturer's instructions. After quality control, RNA-seq libraries were constructed using the BGISEQ500 platform (BGI, China). The raw RNA-seq data were filtered to obtain the clean data, which were used for alignment to the mouse genome (Mus musculus GRCm38.p5, NCBI). Genes with mean fragments per kilobase million lower than 1 in all groups were excluded in subsequent analyses. Statistical significance of differentially expressed genes (DEGs) was calculated based on the raw read counts, with an absolute log2 fold change greater than 1, and adjusted P-value (q-value) less than 0.05. Gene Ontology enrichment analysis was performed using DAVID (https://david.ncifcrf.gov).21,22 The gene expression heatmap was drawn with Heatmapper.2 The results were visualized by the R package ggplot2 (Version 3.6.3; R software).

2.9. Behavioral tests

Animals were habituated to the testing environment for 7 days before behavioral tests. The room temperature (23 ± 1°C) and humidity (60 ± 10%) remained stable for all experiments. For mechanical and thermal sensitivity tests, animals were put under opaque plexiglas chambers on an elevated floor and allowed 30 minutes for habituation before the sensitivity threshold testing. von Frey tests and Hargreaves tests were carried at 2 days before surgery and at 3, 5, 7, 14, 21, and 28 days after surgery during light cycle. Acetone tests were performed 1 day after von Frey tests. Animals were randomly assigned to test groups and the researchers were blinded to animal assignments.

2.9.1. von Frey test

Mechanical allodynia was assessed to calculate 50% paw withdrawal threshold (50% PWT) with the up–down method.5 With a starting force of 0.008g, a series of von Frey hairs with logarithmically incrementing stiffness (0.008-2g; North Coast Medical, Gilroy, CA) were applied to the plantar surface of the hind paw for 2 to 3 seconds. Positive response was determined as sharp withdrawal, shaking, or licking of the limb.

2.9.2. Hargreaves test

Heat hyperalgesia was examined using Hargreaves radiant heat apparatus (IITC Life Science, CA).19 A light beam was focused on the mid-plantar surface of the ipsilateral hind paw. The paw withdrawal latency was recorded. The basal paw withdrawal latency was adjusted to 9 to 12 seconds. The cut off time was 20 seconds to avoid tissue damage.

2.9.3. Acetone test

Cold allodynia was assessed with 20 μL acetone applied to the ventral surface of the ipsilateral hind paw. A camera was used to record the behavior of each mouse from the bottom for 60 seconds. Total duration of acetone-evoked pain-like behaviors (flinching, licking, or biting) was recorded.36

2.10. Statistical analysis

Statistical analysis was performed using GraphPad Prism (Version 8.0.1, CA). Data are presented as mean ± SEM. Two-way analysis of variance with Bonferroni post hoc tests were used for multiple comparisons as appropriate. Each n indicates the number of biologically independent replicates. No statistical methods were used to predetermine sample size. Sample sizes were chosen on the basis of previous experience with similar models. The exact sample size and P value for any given experiment are indicated in the figure legend or in Source Data. Significance was considered at P < 0.05.

3. Results

3.1. Degenerated nucleus pulposus, but not nondegenerated nucleus pulposus, induced chronic sciatica

Normal nondegenerated NP tissues have been used in conventional rat LDH models. To mimic this model in mice, we harvested normal NP tissue from the lumbar spine of donor mice and transplanted them in the left L4/L5 foramen of the acceptor mice to induce L4 nerve root compression (hereafter referred as conventional mouse LDH model, Figs. 1A and B). Axial histological sections of the spinal segment 7 days after transplantation with safranin-O staining confirmed compression of the left L4 nerve root by the transplanted NP tissues, whereas the spinal cord, right L4 nerve root and DRG, and posterior lamina remained structurally intact (Fig. 1A). Pain assessments using von Frey filaments and Hargreaves tests revealed that conventional LDH model mice exhibited mechanical allodynia and heat hyperalgesia. In contrast to the long-lasting pain seen in LDH patients, however, pain in these conventional LDH models lasted for only 7 days, suggesting a transitory pain after acute nerve root compression (combined data are shown in Figs. 1C and D; different sex data are shown in Supplemental Fig. 1, available at https://links.lww.com/PAIN/B662).

F1
Figure 1.:
Establishment of the mouse model of LDH. (A) A representative image showing the axial histological section of the lumbar spine from an LDH model mouse visualized by safranin-O staining. The red arrows indicated that ipsilateral DRG (a) was compressed by NP tissues (b). Vertebral body (c), contralateral DRG (d), spinal cord (e), and posterior lamina (f) are indicated. Scale bar: 500 μm. (B) H&E and safranin-O staining of the nonpunctured and needle-punctured degenerative lumbar discs at day 28 after the surgery. Scale bar: 200 μm. (C–E) Measurements of mechanical allodynia (C), heat hyperalgesia (D), and cold allodynia (E) in male and female conventional and modified LDH models (n = 13-31 mice per group). Values are mean ± SEM. */& P < 0.05, **/&& P < 0.01, ***/&&& P < 0.001 vs sham group, and # P < 0.05, ### P < 0.001, vs conventional LDH model, 2-way analysis of variance with Bonferroni post hoc tests among groups. BL, baseline; DRG, dorsal root ganglia; H&E, hematoxylin and eosin; LDH, lumbar disc herniation; NP, nucleus pulposus; PWL, paw withdrawal latency; PWT, paw withdrawal threshold.

Given the fact that LDH often occurs in patients with disc degeneration, we hypothesized that a disc compression model using degenerated disc may be able to closely mimic the pathology of LDH. Firstly, we induced disc degeneration by needle puncture into the disc. As shown by H&E and safranin-O staining of the disc 4 weeks after puncture, distorted AF, shrunk NP, clustered NP cells, along with narrowed disc height, were observed in the punctured discs, indicating progressive disc degeneration (Fig. 1B). Then, we obtained these degenerated NP tissues (4 weeks after disc puncture) and transferred them into the acceptor mice to compress the L4 nerve root (similar to the conventional LDH model). Strikingly, degenerated NP tissue–resulted compression of the nerve root induced persistent pain (sciatica) as shown by mechanical allodynia and heat hyperalgesia in the ipsilateral hind limb even 28 days after the surgery in both male and female mice (combined data are shown in Figs. 1C and D; different sex data are shown in Supplemental Fig. 1, available at https://links.lww.com/PAIN/B662). In addition, only degenerated, but not normal, NP tissue-caused nerve root compression induced cold allodynia in mice, albeit within only 1 week (≤6 days, Fig. 1E). Together, these data demonstrated that compression of the nerve root with degenerated NP, rather than normal NP, mimicked the chronic process of clinical LDH with sustained sciatica. We hereby refer this model to modified LDH model.

3.2. Innate immune cell responses in the dorsal root ganglia and spinal dorsal horn after lumbar disc herniation modeling

The main differences between the conventional and our modified LDH model lie in the NP tissues used. It has been well-recognized that disc degeneration is usually accompanied by chronic inflammation within the tissue, characterized by macrophage infiltration and cytokine release. It is likely that, other than mechanical compression, transferred degenerated NP tissue-elicited inflammation contributed to the persistent pain in the modified LDH models. To test this hypothesis, we investigated the changes of innate immune cells in the disc, DRG, and SDH. We observed a prominent population of macrophages (F4/80+ cells) in the needle-punctured degenerated discs, but not in the normal control discs, indicating macrophage infiltration into the degenerated discs (Fig. 2A). Interestingly, compared with sham mice, macrophages were increased in the L4 DRG 1 week after transplantation in both conventional and modified LDH model mice (P < 0.01 for both). However, at the end of the second week, the number of macrophages went back to normal in the conventional LDH models, whereas it remained high in the modified models (P < 0.01), suggesting a prolonged infiltration upon degenerated NP transplantation (Figs. 2B and C), which can also be observed in female LDH mice model (Supplemental Fig. 2A, available at https://links.lww.com/PAIN/B662). By 28 days, the density of macrophage in the DRG returned to basal level in all groups (Figs. 2B and C). As microglia in SDH play an important role in pain regulation, we further investigated microgliosis in these mice. Notably, significant activation of microglia characterized by morphological changes and increased numbers in the ipsilateral L4 SDH was only observed in modified, but not conventional, LDH models (both male and female mice) at day 7 postsurgery (Fig. 3; Supplemental Fig. 2B, available at https://links.lww.com/PAIN/B662). The increased microglia number in the modified LDH models returned to basal level 14 days after the surgery (Fig. 3). These data indicated that degenerated discs may trigger prolonged and robust immune responses as shown by the substantial and extended macrophage infiltration in degenerated discs and DRG, along with microglia activation in the SDH in the modified LDH model mice.

F2
Figure 2.:
Increased number of macrophages in the intervertebral disc and DRG after LDH modeling. (A) Representative images of macrophages (F4/80+) in the intervertebral disc at 7 days after disc puncture. Scale bar: 100 μm. (B) Representative immunolabeling of F4/80+ macrophages in both conventional and modified LDH model groups in the L4 DRG at 7, 14, and 28 days after the surgery. Scale bar: 100 μm for outer panels and 50 μm for inner panels. (C) Quantitative analysis of macrophages in DRG at 7, 14 and 28 days after the surgery (n = 5-7 mice per group). Values are mean ± SEM. ** P < 0.01, *** P < 0.001, 2-way analysis of variance with Bonferroni post hoc tests among groups. LDH, lumbar disc herniation.
F3
Figure 3.:
Activation of microglia in SDH after LDH modeling. (A) Iba1+ microglia significantly activated and accumulated in L4 SDH at day 7 postsurgery in modified LDH model mice, but not sham and conventional LDH model mice. Scale bar: 100 μm for outer panels and 10 μm for inner panels. Dotted lines indicate the superficial region of SDH. (B) Density of SDH microglia at 7, 14, and 28 days after the surgery (n = 4-5 mice per group). Values are mean ± SEM. ** P < 0.01, *** P < 0.001, 2-way analysis of variance with Bonferroni post hoc tests among groups. LDH, lumbar disc herniation; SDH, spinal dorsal horn.

3.3. Persistent ablation of macrophages and microglia prevents lumbar disc herniation-induced sciatica

Both microglia and macrophages play important roles in regulating pain. To investigate whether macrophage infiltration and microglia activation contribute to chronic sciatica in the modified LDH models, we have used pharmacological approach to ablate macrophages and microglia. Mice were fed with chow containing PLX5622, a selective antagonist of CSF1R that selectively kill microglia and macrophages, 7 days before the surgery until the end of pain evaluation (schematically shown in Fig. 4A). Macrophages in DRG and transplanted NP tissues, as well as microglia in SDH, were almost completely eliminated after continuous treatment of PLX5622 (Figs. 4B–D; Supplemental Fig. 2, available at https://links.lww.com/PAIN/B662). Both male and female mice fed with PLX5622 and control diet showed no difference in baseline mechanical and thermal sensitivities, indicating that the absence of microglia and macrophages did not affect basal nociception. However, mechanical allodynia and thermal hyperalgesia were significantly reduced in the mice fed with PLX5622 compared with those fed with control diet (combined data are shown in Figs. 4E–G; different sex data are shown in Supplemental Fig. 3, available at https://links.lww.com/PAIN/B662). Findings suggested that the activation of macrophages and microglia was an important trigger for the development and maintenance of LDH-induced sciatica.

F4
Figure 4.:
Persistent ablation of macrophages and microglia prevents LDH-induced sciatica. (A) Experimental schedule for the treatment of PLX5622 and behavioral tests. (B–D) Microglia in SDH (B) and macrophages in DRG (C) and transplanted NP tissues (D) was eliminated by continuous treatment of PLX5622 for 29 days. (E–G) Compared with the control group, ablation of microglia and macrophages significantly attenuates mechanical allodynia (E), heat hyperalgesia (F), and cold allodynia (G) in male and female modified LDH model mice (n = 9-17 mice per group). Values are mean ± SEM. *** P < 0.001 vs control diet group, 2-way analysis of variance with Bonferroni post hoc tests among groups. BL, baseline; LDH, lumbar disc herniation; NP, nucleus pulposus; PO, preoperation; PWT, paw withdrawal threshold; PWL, paw withdrawal latency; SDH, spinal dorsal horn.

3.4. Macrophages and microglia were essential to maintain lumbar disc herniation;-induced mechanical allodynia

After discontinuation of PLX5622, macrophages and microglia can rapidly repopulate and return to normal levels to exert biological functions.24 To explore the roles of repopulated macrophages and microglia in the maintenance of LDH-induced sciatica, mice were kept on chow feed containing PLX5622 for 7 days before surgery, and then PLX5622 was replaced with control feed at 0, 7, and 14 days after the surgery (schematically shown in Fig. 5A). Compared with control group, mechanical allodynia in the modified LDH model mice was alleviated when treated with PLX5622, but reoccurred when PLX5622 was replaced with control diets at day 7 and 14 postsurgery (Fig. 5B). For Hargreaves tests, however, there was no significant difference in heat hyperalgesia for LDH model mice with PLX5622 discontinued on the day of surgery until postoperative day 14. After PLX5622 replacement at day 7 and 14, however, heat hyperalgesia did not reoccur (Fig. 5C). In addition, early cold allodynia was significantly alleviated in all PLX5622-treated LDH model mice (Fig. 5D). Results suggested that macrophage and microglia were necessary for maintaining LDH-induced mechanical allodynia. Yet, only when macrophages and microglia were ablated at the initial stage of LDH modeling can LDH-induced heat hyperalgesia be persistently alleviated.

F5
Figure 5.:
Macrophages and microglia play a key role in the maintenance of LDH-induced sciatica. (A) Experimental schedule for PLX5622 treatment and behavioral tests. (B) Mechanical allodynia was alleviated when treated with PLX5622 but reoccurred when PLX5622 was replaced with control diet at the day 7 and 14, respectively. (C, D) Heat hyperalgesia (C) and cold allodynia (D) were significantly alleviated regardless of PLX5622 treatment or replacement in modified LDH model mice (n = 8-9 mice per group). Values are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, 2-way analysis of variance with Bonferroni post hoc tests among groups. BL, baseline; LDH, lumbar disc herniation; PO, preoperation; PWT, paw withdrawal threshold; PWL, paw withdrawal latency.

3.5. Transcriptional analysis of dorsal root ganglia in modified lumbar disc herniation mice model

As the modified LDH model successfully induced persistent sciatic pain in mice, we used male mice model to investigate DEGs involved in chronic sciatica. Transcriptomic analyses revealed that 71 genes (66 upregulated and 5 downregulated) and 35 genes (34 upregulated and 1 downregulated) were differentially expressed in DRG at day 7 (shown in Supplemental Table 1, available at https://links.lww.com/PAIN/B662) and day 14 (shown in Supplemental Table 2, available at https://links.lww.com/PAIN/B662) after degenerated NP tissue compression, respectively. Among them, 17 of 89 DEGs, including 16 upregulated and 1 downregulated, were overlapped (Fig. 6A). Gene Ontology enrichment analysis revealed that 83 upregulated DEGs (both day 7 and 14) were enriched in multiple immune responses and inflammation regulations (Fig. 6B), suggesting a key role of immune responses in the development of LDH-induced sciatica. Furthermore, 7 of 66 (10.6%) upregulated DEGs at day 7, and 7 of 34 (20.6%) upregulated DEGs at day 14, were related to macrophage functions (Fig. 6C). In addition, 6 of the overlapped 17 (35.3%) DEGs, including mmp12, trem2, gpnmb, atf3, fyb, and cd68, which persistently upregulated at both day 7 and 14, were also associated with macrophage functions (Figs. 6D and E). Immunofluorescence staining confirmed that CD68 and TREM2 were upregulated in F4/80+ macrophages of ipsilateral DRG 7 days after nerve root compression with degenerated NP (Figs. 7A and B). ATF3 and CSF1 were upregulated in ipsilateral DRG neurons (Fig. 7C), indicating that nerve injury was induced by degenerated NP compression. Correspondingly, a significant increase of CSF1R+ microglia was observed in the ipsilateral SDH (Fig. 7D). Collectively, these data suggested that degenerated NP compression induced significant macrophage-related immune responses in DRG, which may deeply engage in the development and maintenance of LDH-induced sciatica.

F6
Figure 6.:
RNA-seq analysis of the DRG tissues from the modified LDH models. (A) Venn diagram of DEGs in DRG at day 7 and 14 after modified LDH modeling, as compared with sham group. (B) Gene Ontology enrichment analysis of all DEGs at day 7 and 14 after modified LDH modeling. (C) Heat map of macrophage-related DEGs at day 7 and 14 after modified LDH modeling. (D–E) Volcano maps of DEGs at day 7 (D) and day 14 (E) after modified LDH modeling. Macrophage-related genes were marked (n = 5 male mice per group). DEGs, differentially expressed genes; LDH, lumbar disc herniation; RNA-seq, RNA sequencing.
F7
Figure 7.:
Changes of DRG macrophages and SDH microglia after LDH modeling. (A–B) CD68 (A) and TREM2 (B) were upregulated in F4/80+ macrophages of ipsilateral DRG at day 7 postsurgery in male modified LDH model mice but not in DRG of sham group. Scale bar: 100 μm for outer panels and 10 μm for inner panels. (C) Co-expression of CSF1 and ATF3 in DRG neurons 7 days post modified LDH modeling compared with DRG of sham group. Scale bar: 100 μm for outer panels and 10 μm for inner panels. (D) CSF1R+ Iba1+ microglia significantly activated and accumulated in ipsilateral L4 SDH at day 7 postsurgery in male modified LDH model mice but not in SDH of sham group. Scale bar: 200 μm for outer panels and 20 μm for inner panels. Dotted lines indicate the superficial region of SDH. CSF1R, colony-stimulating factor 1 receptor; LDH, lumbar disc herniation; SDH, spinal dorsal horn.

4. Discussion

Using NP tissues from degenerated discs, the current study successfully established a modified model of LDH to induce persistent sciatica in mice. Compression of the nerve root with nondegenerated NP tissues (the conventional LDH model) only led to transient mechanical allodynia and heat hyperalgesia, with temporary infiltration of macrophages in the DRG and mild activation of microglia in the spinal cord. Instead, nerve root compression using degenerated NP tissues (the modified LDH model) can initiate remarkable and persistent mechanical allodynia and heat hyperalgesia, with prolonged and increased macrophage infiltration in DRG and significant microglia activation in the spinal cord. Moreover, persistent ablation of macrophages and microglia using PLX5622 prevented mechanical allodynia and thermal hyperalgesia in the modified LDH model mice. In addition, mechanical allodynia reoccurred 7 days after the replacement of PLX5622, along with repopulation of macrophages and microglia. Using RNA-seq analysis, the current study depicted transcriptional profile of DRG and identified several continuously upregulated macrophage-related genes, including mmp12, trem2, gpnmb, atf3, and fyb, as candidate targets for future study of LDH-induced chronic sciatica. Our results suggested that macrophages and microglia play an essential role in the development and maintenance of LDH-induced sciatica.

4.1. Establishment of a modified lumbar disc herniation model in mice

A reliable animal model is fundamental for understanding the neurobiology of LDH-induced sciatica and developing novel therapeutic strategies. Using allogeneic degenerated NP tissues as a nerve root compressor, the current study successfully established a modified LDH model that can induce persistent sciatica in mice. Although a variety of animal models of LDH have been developed over decades, including rats, sheep, pigs, dogs, and rabbits,15,48 those models used nondegenerated normal discs or stainless steel for nerve root compression, were different from LDH patients in whom the herniated NP tissues have been undergoing a long procession of degeneration.42,50,61 Compared with previous models with limited pain duration,64,68 degenerated disc and NP tissues, as induced by needle puncture, can induce profound mechanical allodynia and heat hyperalgesia in mice. Such findings are in accordance with clinical observations that some LDH patients suffered from chronic sciatica and severe disc degeneration typically require surgical interventions or even fail to respond to discectomy.37,60 This newly developed mouse model of LDH can reliably induce long-lasting sciatica and, therefore, can be used as a tool for further study of LDH-induced sciatica.

4.2. Macrophages and microglia in sciatica

Emerging evidence suggests that neuron–immune interactions play a pivotal role in the initiation and maintenance of neuropathic pain in both PNS and CNS.10,55 The present study revealed that there was significant infiltration of macrophages in degenerated discs and DRG, and substantial activation of microglia in the spinal cord after LDH modeling using degenerated NP tissues, which may be attributed to mechanical compression and local inflammatory responses. There is now considerable consensus that macrophages and microglia are important contributors to chronic pain.28,56,66 In disc herniation, the originally immune-privileged NP tissues exposed to immune system and triggered a cascade of immune responses. Regarded as a major source of inflammation, neutrophils and monocytes/macrophages can quickly infiltrate into the herniated disc tissues and ignite a harsh pro-inflammatory response. Moreover, the interactions between macrophages and NP cells may further promote the production of pro-inflammatory factors and the degradation of extracellular matrix, resulting in persistent chemical irritations in the nearby DRG.4,41 It is reported that LDH-induced pain hypersensitivity can be effectively alleviated by blocking the infiltration and activation of macrophages in DRG.68 In addition, nerve injury was induced by nerve root compression with degenerated NP tissues, along with significant upregulation of ATF3 and CSF1 in DRG neurons. Spinal microglia can rapidly respond to peripheral stimuli through upregulating molecules, such as CSF1, Nox2, P2Y12, and P2X4, and releasing mediators such as TNF-α and BDNF.3,30 Although mechanisms of neuronal excitation by inflammatory signals in both PNS and CNS have been investigated,37,67 the interactions of macrophages and DRG neurons largely remain unstudied in LDH-induced sciatica.

In the conventional LDH model, the inflammation induced by the undegenerated NP tissues was not as severe as that of degenerated NP, which explains the transient pain behaviors induced.27,53,58 Consistent with our modified mouse model of LDH, combination of chemical irritation and mechanical compression can induce persistent pain hypersensitivity, along with the enhanced reactivities of astrocyte and microglia in SDH.53 In addition, not only did we demonstrate that preventive ablation of macrophage and microglia alleviated LDH induced sciatica but also found that the mechanical allodynia, but not heat hyperalgesia, reoccurred after the repopulation of macrophages and microglia, as supported by previous studies.51,66 It is likely that the upregulation of inflammatory cytokines, such as IL-1β and TNF-α in both DRG and spinal cord, which mainly derived from macrophages and microglia, is implicated in the central sensitization process.32 Although such correlation between pain maintenance and immune cell responses remains unclear, the current study provided a rationale for targeting both macrophage and microglia to prevent the development of LDH-induced chronic pain.

4.3. Potential targets for treating chronic sciatica

Although there are numerous therapeutic strategies available for treating LDH, chronic and recurrent sciatica remains to be clinical challenges.39,57 Using RNA-seq analysis, the current study identified some macrophage-related DEGs, including mmp12, trem2, gpnmb, atf3, cd68, and fyb, which were continuously upregulated in DRG after LDH modeling using degenerated NP tissues. Matrix metallopeptidase 12 (MMP12), enabling macrophage infiltration into injured peripheral nerves by degrading the extracellular matrix,25 was implicated in the pathogenesis of several inflammatory, vascular, and neurological diseases.6 Local administration of its selective inhibitor alleviated sciatic nerve ligation–induced mechanical allodynia and thermal hyperalgesia.34 Although TREM-2 (triggering receptor expressed on myeloid cells 2) signaling was often portrayed as anti-inflammatory and reparative, other studies indicated that TREM-2 may participate in a feed forward mechanism to amplify the accumulation of macrophages.59,62 Recent study has shown that GPNMB (glycoprotein nonmetastatic melanoma protein B) was expressed in microglia and macrophage,23 and its activation plays an important role in nociceptive processes by modulating inflammatory responses.20 With the understanding of sciatica evolving from solely neuronal mechanisms to neuron–immune and neuron–glial interactions, our RNA-seq analysis results may provide clues for novel therapeutic targets for LDH-induced sciatica.

4.4. Unanswered questions and future research

There are some shortcomings in this study. Firstly, it is important to elucidate the roles of macrophages and microglia in LDH-induced sciatica separately. Secondly, multiple inflammatory factors were not evaluated for both degenerated discs and insulted nerve roots and DRG in the current study, although previous studies have reported that a cascade of immune responses were involved in disc degeneration and LDH. Thirdly, except for behavioral tests solely dependent on reflex responses, more approaches, including measurements for spontaneous pain,7 are required to validate the modified LDH model in this study. Although many macrophage-related genes were found persistently upregulated in DRG of LDH model mice, the mechanisms of neuron–immune interactions in LDH-induced sciatica remain to be studied.

5. Conclusions

In conclusion, this study established and modified the mouse model of LDH to induce persistent sciatica. Increased infiltration of macrophages in DRG and activation of microglia in spinal cord are essential for the initiation and maintenance of LDH-induced sciatica. Targeting macrophages and microglia and their interactions with sensory neurons may be a possible treatment for chronic sciatica.

Conflict of interest statement

The authors have no conflicts of interest to declare.

Appendix A. Supplemental digital content

Supplemental digital content associated with this article can be found online at https://links.lww.com/PAIN/B662.

Supplemental video content

A video abstract associated with this article can be found at https://links.lww.com/PAIN/B663.

Acknowledgements

This work was supported by National Natural Science Foundation of China (81772382 and 32070974), Science Technology Department of Zhejiang Province (2020C03042), the Fundamental Research Funds for the Central Universities of China (2019FZA7009), and the Central Universities granted by Zhejiang University (No. 2021FZZX005-29). PLX5622 was provided without financial support under a Materials Transfer Agreement with Plexxikon Inc, Berkeley, CA.

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Keywords:

Lumbar disc herniation; Pain; Sciatica; Macrophage; Microglia

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