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Autologous nucleus pulposus implanted into lumbar subchondral bone to create an animal model of Modic changes

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The establishment of animal models of modic change (MC) is an important basis for studying MC. Fifty-four New Zealand White rabbits were divided into sham-operation group, muscle implantation group (ME group) and nucleus pulposus implantation group (NPE group). In the NPE group, the intervertebral disc was exposed by anterolateral lumbar surgical approach and a needle was used to puncture the L5 vertebral body near the end plate. NP was extracted from the L1/2 intervertebral disc by a syringe and injected into it. Drilling a hole in the subchondral bone. The surgical procedures and drilling methods in the muscle implantation group and the sham-operation group were the same as those in the NP implantation group. In the ME group, a piece of muscle was placed into the hole, while in the sham-operation group, nothing was placed into the hole. After the operation, MRI scanning and molecular biological testing were performed. The signal in the NPE group changed, but there was no obvious signal change in the sham-operation group and the ME group. Histological observation showed that abnormal tissue proliferation was observed at the implantation site, and the expression of IL-4, IL-17 and IFN-γ was increased in the NPE group. Implantation of NP into the subchondral bone can form an animal model of MC.
Modic changes (MC) are lesions of the vertebral endplates and adjacent bone marrow visible on magnetic resonance imaging (MRI). They are quite common in individuals with associated symptoms1. Many studies have emphasized the importance of MC due to its association with low back pain (LBP)2,3. de Roos et al.4 and Modic et al.5 independently first described three different types of subchondral signal abnormalities in the vertebral bone marrow. Modic type I changes are hypointense on T1-weighted (T1W) sequences and hyperintense on T2-weighted (T2W) sequences. This lesion reveals fissure endplates and adjacent vascular granulation tissue in the bone marrow. Modic type II changes show high signal on both T1W and T2W sequences. In this type of lesion, endplate destruction can be found, as well as histological fatty replacement of the adjacent bone marrow. Modic type III changes show low signal in T1W and T2W sequences. Sclerotic lesions corresponding to the endplates have been observed6. MC is considered a pathological disease of the spine and is closely associated with many degenerative diseases of the spine7,8,9.
Considering the available data, several studies have provided detailed insights into the etiology and pathological mechanisms of MC. Albert et al. suggested that MC may be caused by disc herniation8. Hu et al. attributed MC to severe disc degeneration10. Kroc proposed the concept of “internal disc rupture,” which states that repetitive disc trauma may lead to microtears in the endplate. After cleft formation, endplate destruction by the nucleus pulposus (NP) may trigger an autoimmune response, which further leads to the development of MC11. Ma et al. shared a similar view and reported that NP-induced autoimmunity plays a key role in the pathogenesis of MC12.
Immune system cells, especially CD4+ T helper lymphocytes, play a critical role in the pathogenesis of autoimmunity13. The recently discovered Th17 subset produces the proinflammatory cytokine IL-17, promotes chemokine expression, and stimulates T cells in damaged organs to produce IFN-γ14. Th2 cells also play a unique role in the pathogenesis of immune responses. Expression of IL-4 as a representative Th2 cell can lead to severe immunopathological consequences15.
Although many clinical studies have been conducted on MC16,17,18,19,20,21,22,23,24, there is still a lack of suitable animal experimental models that can mimic the MC process that frequently occurs in humans and can be used to investigate the etiology or new treatments such as targeted therapy. To date, only a few animal models of MC have been reported to study the underlying pathological mechanisms.
Based on the autoimmune theory proposed by Albert and Ma, this study established a simple and reproducible rabbit MC model by autotransplanting NP near the drilled vertebral end plate. Other objectives are to observe the histological characteristics of the animal models and evaluate the specific mechanisms of NP in the development of MC. To this end, we use techniques such as molecular biology, MRI, and histological studies to study the progression of MC.
Two rabbits died of bleeding during surgery, and four rabbits died during anesthesia during MRI. The remaining 48 rabbits survived and showed no behavioral or neurological signs after surgery.
MRI shows that the signal intensity of the embedded tissue in different holes is different. The signal intensity of the L5 vertebral body in the NPE group gradually changed at 12, 16 and 20 weeks after insertion (T1W sequence showed low signal, and T2W sequence showed mixed signal plus low signal) (Fig. 1C), while the MRI appearances of the other two groups of embedded parts remained relatively stable during the same period (Fig. 1A, B).
(A) Representative sequential MRIs of the rabbit lumbar spine at 3 time points. No signal abnormalities were found in the images of the sham-operation group. (B) The signal characteristics of the vertebral body in the ME group are similar to those in the sham-operation group, and no significant signal change is observed at the embedding site over time. (C) In the NPE group, the low signal is clearly visible in the T1W sequence, and the mixed signal and low signal are clearly visible in the T2W sequence. From the 12-week period to the 20-week period, the sporadic high signals surrounding the low signals in the T2W sequence decrease.
Obvious bone hyperplasia can be seen at the implantation site of the vertebral body in the NPE group, and the bone hyperplasia occurs faster from 12 to 20 weeks (Fig. 2C) compared with the NPE group, no significant change is observed in the modeled vertebral bodies; Sham group and ME group (Fig. 2C) 2A,B).
(A) The surface of the vertebral body at the implanted portion is very smooth, the hole heals well, and there is no hyperplasia in the vertebral body. (B) The shape of the implanted site in the ME group is similar to that in the sham operation group, and there is no obvious change in the appearance of the implanted site over time. (C) Bone hyperplasia occurred at the implanted site in the NPE group. The bone hyperplasia increased rapidly and even extended through the intervertebral disc to the contralateral vertebral body.
Histological analysis provides more detailed information about bone formation. Figure 3 shows the photographs of the postoperative sections stained with H&E. In the sham-operation group, the chondrocytes were well arranged and no cell proliferation was detected (Fig. 3A). The situation in the ME group was similar to that in the sham-operation group (Fig. 3B). However, in the NPE group, a large number of chondrocytes and proliferation of NP-like cells were observed at the implantation site (Fig. 3C);
(A) Trabeculae can be seen near the end plate, the chondrocytes are neatly arranged with uniform cell size and shape and no proliferation (40 times). (B) The condition of the implantation site in the ME group is similar to that of the sham group. Trabeculae and chondrocytes can be seen, but there is no obvious proliferation at the implantation site (40 times). (B) It can be seen that chondrocytes and NP-like cells proliferate significantly, and the shape and size of chondrocytes are uneven (40 times).
The expression of interleukin 4 (IL-4) mRNA, interleukin 17 (IL-17) mRNA, and interferon γ (IFN-γ) mRNA were observed in both the NPE and ME groups. When the expression levels of target genes were compared, the gene expressions of IL-4, IL-17, and IFN-γ were significantly increased in the NPE group compared with those of the ME group and the sham operation group (Fig. 4) (P < 0.05). Compared with the sham operation group, the expression levels of IL-4, IL-17, and IFN-γ in the ME group increased only slightly and did not reach statistical change (P > 0.05).
The mRNA expression of IL-4, IL-17 and IFN-γ in the NPE group showed a significantly higher trend than those in the sham operation group and the ME group (P < 0.05).
In contrast, the expression levels in the ME group showed no significant difference (P>0.05).
Western blot analysis was performed using commercially available antibodies against IL-4 and IL-17 to confirm the altered mRNA expression pattern. As shown in Figures 5A,B, compared with the ME group and the sham operation group, the protein levels of IL-4 and IL-17 in the NPE group were significantly increased (P < 0.05). Compared with the sham operation group, the protein levels of IL-4 and IL-17 in the ME group also failed to reach statistically significant changes (P> 0.05).
(A) The protein levels of IL-4 and IL-17 in the NPE group were significantly higher than those in the ME group and placebo group (P < 0.05). (B) Western blot histogram.
Due to the limited number of human samples obtained during surgery, clear and detailed studies on the pathogenesis of MC are somewhat difficult. We attempted to establish an animal model of MC to study its potential pathological mechanisms. At the same time, radiological evaluation, histological evaluation and molecular biological evaluation were used to follow the course of MC induced by NP autograft. As a result, the NP implantation model resulted in a gradual change in signal intensity from 12-week to 20-week time points (mixed low signal in T1W sequences and low signal in T2W sequences), indicating tissue changes, and the histological and molecular biological evaluations confirmed the results of the radiological study.
The results of this experiment show that visual and histological changes occurred at the site of vertebral body infringement in the NPE group. At the same time, the expression of IL-4, IL-17 and IFN-γ genes, as well as IL-4, IL-17 and IFN-γ were observed, indicating that the infringement of autologous nucleus pulposus tissue in the vertebral body may cause a series of signal and morphological changes. It is easy to find that the signal characteristics of the vertebral bodies of the animal model (low signal in the T1W sequence, mixed signal and low signal in the T2W sequence) are very similar to those of human vertebral cells, and the MRI characteristics also confirm the observations of histology and gross anatomy, that is, the changes in the vertebral body cells are progressive. Although the inflammatory response caused by acute trauma may appear soon after puncture, MRI results showed that progressively increasing signal changes appeared 12 weeks after puncture and persisted up to 20 weeks without any signs of recovery or reversal of MRI changes. These results suggest that autologous vertebral NP is a reliable method for establishing progressive MV in rabbits.
This puncture model requires adequate skill, time, and surgical effort. In preliminary experiments, dissection or excessive stimulation of the paravertebral ligamentous structures may result in the formation of vertebral osteophytes. Care should be taken not to damage or irritate the adjacent discs. Since the depth of penetration must be controlled to obtain consistent and reproducible results, we manually made a plug by cutting off the sheath of a 3 mm long needle. Using this plug ensures uniform drilling depth in the vertebral body. In preliminary experiments, three orthopedic surgeons involved in the operation found 16-gauge needles easier to work with than 18-gauge needles or other methods. To avoid excessive bleeding during drilling, holding the needle still for a while will provide a more suitable insertion hole, suggesting that a certain degree of MC can be controlled in this way.
Although many studies have targeted MC, little is known about the etiology and pathogenesis of MC25,26,27. Based on our previous studies, we found that autoimmunity plays a key role in the occurrence and development of MC12. This study examined the quantitative expression of IL-4, IL-17, and IFN-γ, which are the main differentiation pathways of CD4+ cells after antigen stimulation. In our study, compared with the negative group, the NPE group had higher expression of IL-4, IL-17, and IFN-γ, and the protein levels of IL-4 and IL-17 were also higher.
Clinically, IL-17 mRNA expression is increased in NP cells from patients with disc herniation28. Increased IL-4 and IFN-γ expression levels were also found in an acute non-compressive disc herniation model compared with healthy controls29. IL-17 plays a key role in inflammation, tissue injury in autoimmune diseases30 and enhances the immune response to IFN-γ31. Enhanced IL-17-mediated tissue injury has been reported in MRL/lpr mice32 and autoimmunity-susceptible mice33. IL-4 can inhibit the expression of proinflammatory cytokines (such as IL-1β and TNFα) and macrophage activation34. It was reported that the mRNA expression of IL-4 was different in the NPE group compared to IL-17 and IFN-γ at the same time point; The mRNA expression of IFN-γ in the NPE group was significantly higher than that in the other groups. Therefore, IFN-γ production may be a mediator of the inflammatory response induced by NP intercalation. Studies have shown that IFN-γ is produced by multiple cell types, including activated type 1 helper T cells, natural killer cells, and macrophages35,36, and is a key pro-inflammatory cytokine that promotes immune responses37.
This study suggests that autoimmune response may be involved in the occurrence and development of MC. Luoma et al. found that the signal characteristics of MC and prominent NP are similar on MRI, and both show high signal in T2W sequence38. Some cytokines have been confirmed to be closely associated with the occurrence of MC, such as IL-139. Ma et al. suggested that the upward or downward protrusion of NP may have a great influence on the occurrence and development of MC12. Bobechko40 and Herzbein et al.41 reported that NP is an immunotolerant tissue that cannot enter the vascular circulation from birth. NP protrusions introduce foreign bodies into the blood supply, thereby mediating local autoimmune reactions42. Autoimmune reactions can induce a large number of immune factors, and when these factors are continuously exposed to tissues, they can cause changes in signaling43. In this study, overexpression of IL-4, IL-17 and IFN-γ are typical immune factors, further proving the close relationship between NP and MCs44. This animal model well mimics the NP breakthrough and entry into the end plate. This process further revealed the impact of autoimmunity on MC.
As expected, this animal model provides us with a possible platform to study MC. However, this model still has some limitations: firstly, during the animal observation phase, some intermediate-stage rabbits need to be euthanized for histological and molecular biology testing, so some animals “fall out of use” over time. Secondly, although three time points are set in this study, unfortunately, we only modeled one type of MC (Modic type I change), so it is not enough to represent the human disease development process, and more time points need to be set to better observe all the signal changes. Thirdly, the changes in tissue structure can indeed be clearly shown by histological staining, but some specialized techniques can better reveal the microstructural changes in this model. For example, polarized light microscopy was used to analyze the formation of fibrocartilage in rabbit intervertebral discs45. The long-term effects of NP on MC and endplate require further study.
Fifty-four male New Zealand white rabbits (weight about 2.5-3 kg, age 3-3.5 months) were randomly divided into sham operation group, muscle implantation group (ME group) and nerve root implantation group (NPE group). All experimental procedures were approved by the Ethics Committee of Tianjin Hospital, and the experimental methods were carried out in strict accordance with the approved guidelines.
Some improvements have been made to the surgical technique of S. Sobajima 46 . Each rabbit was placed in a lateral recumbency position and the anterior surface of five consecutive lumbar intervertebral discs (IVDs) was exposed using a posterolateral retroperitoneal approach. Each rabbit was given general anesthesia (20% urethane, 5 ml/kg via the ear vein). A longitudinal skin incision was made from the lower edge of the ribs to the pelvic brim, 2 cm ventral to the paravertebral muscles. The right anterolateral spine from L1 to L6 was exposed by sharp and blunt dissection of the overlying subcutaneous tissue, retroperitoneal tissue, and muscles (Fig. 6A). The disc level was determined using the pelvic brim as an anatomical landmark for the L5-L6 disc level. Use a 16-gauge puncture needle to drill a hole near the end plate of the L5 vertebra to a depth of 3 mm (Fig. 6B). Use a 5-ml syringe to aspirate the autologous nucleus pulposus in the L1-L2 intervertebral disc (Fig. 6C). Remove the nucleus pulposus or muscle according to the requirements of each group. After the drill hole is deepened, absorbable sutures are placed on the deep fascia, superficial fascia and skin, taking care not to damage the periosteal tissue of the vertebral body during surgery.
(A) The L5–L6 disc is exposed via a posterolateral retroperitoneal approach. (B) Use a 16-gauge needle to drill a hole near the L5 endplate. (C) Autologous MFs are harvested.
General anesthesia was administered with 20% urethane (5 ml/kg) administered via the ear vein, and lumbar spine radiographs were repeated at 12, 16, and 20 weeks postoperatively.
Rabbits were sacrificed by intramuscular injection of ketamine (25.0 mg/kg) and intravenous sodium pentobarbital (1.2 g/kg) at 12, 16 and 20 weeks after surgery. The entire spine was removed for histological analysis and real analysis was performed. Quantitative reverse transcription (RT-qPCR) and Western blotting were used to detect changes in immune factors.
MRI examinations were performed in rabbits using a 3.0 T clinical magnet (GE Medical Systems, Florence, SC) equipped with an orthogonal limb coil receiver. Rabbits were anesthetized with 20% urethane (5 mL/kg) via the ear vein and then placed supine within the magnet with the lumbar region centered on a 5-inch diameter circular surface coil (GE Medical Systems). Coronal T2-weighted localizer images (TR, 1445 ms; TE, 37 ms) were acquired to define the location of the lumbar disc from L3–L4 to L5–L6. Sagittal plane T2-weighted slices were acquired with the following settings: fast spin-echo sequence with a repetition time (TR) of 2200 ms and an echo time (TE) of 70 ms, matrix; visual field of 260 and eight stimuli; The cutting thickness was 2 mm, the gap was 0.2 mm.
After the last photograph was taken and the last rabbit was killed, the sham, muscle-embedded, and NP discs were removed for histological examination. Tissues were fixed in 10% neutral buffered formalin for 1 week, decalcified with ethylenediaminetetraacetic acid, and paraffin sectioned. Tissue blocks were embedded in paraffin and cut into sagittal sections (5 μm thick) using a microtome. Sections were stained with hematoxylin and eosin (H&E).
After collecting the intervertebral discs from the rabbits in each group, total RNA was extracted using a UNIQ-10 column (Shanghai Sangon Biotechnology Co., Ltd., China) according to the manufacturer’s instructions and an ImProm II reverse transcription system (Promega Inc., Madison, WI, USA). Reverse transcription was performed.
RT-qPCR was performed using a Prism 7300 (Applied Biosystems Inc., USA) and SYBR Green Jump Start Taq ReadyMix (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The PCR reaction volume was 20 μl and contained 1.5 μl of diluted cDNA and 0.2 μM of each primer. Primers were designed by OligoPerfect Designer (Invitrogen, Valencia, CA) and manufactured by Nanjing Golden Stewart Biotechnology Co., Ltd. (China) (Table 1). The following thermal cycling conditions were used: initial polymerase activation step at 94°C for 2 min, then 40 cycles of 15 s each at 94°C for template denaturation, annealing for 1 min at 60°C, extension, and fluorescence. measurements were performed for 1 min at 72°C. All samples were amplified three times and the average value was used for RT-qPCR analysis. Amplification data were analyzed using FlexStation 3 (Molecular Devices, Sunnyvale, CA, USA). IL-4, IL-17, and IFN-γ gene expression were normalized to the endogenous control (ACTB). Relative expression levels of target mRNA were calculated using the 2-ΔΔCT method.
Total protein was extracted from tissues using a tissue homogenizer in RIPA lysis buffer (containing a protease and phosphatase inhibitor cocktail) and then centrifuged at 13,000 rpm for 20 min at 4°C to remove tissue debris. Fifty micrograms of protein were loaded per lane, separated by 10% SDS-PAGE, and then transferred to a PVDF membrane. Blocking was performed in 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 h at room temperature. The membrane was incubated with rabbit anti-decorin primary antibody (diluted 1:200; Boster, Wuhan, China) (diluted 1:200; Bioss, Beijing, China) overnight at 4°C and reacted on the second days; with secondary antibody (goat anti-rabbit immunoglobulin G at 1:40,000 dilution) combined with horseradish peroxidase (Boster, Wuhan, China) for 1 hour at room temperature. Western blot signals were detected by increased chemiluminescence on the chemiluminescent membrane after X-ray irradiation. For densitometric analysis, blots were scanned and quantified using BandScan software and the results were expressed as the ratio of target gene immunoreactivity to tubulin immunoreactivity.
Statistical calculations were performed using the SPSS16.0 software package (SPSS, USA). Data collected during the study were expressed as mean ± standard deviation (mean ± SD) and analyzed using one-way repeated measures analysis of variance (ANOVA) to determine differences between the two groups. P < 0.05 was considered statistically significant.
Thus, the establishment of an animal model of MC by implanting autologous NPs into the vertebral body and performing macroanatomical observation, MRI analysis, histological evaluation and molecular biological analysis may become an important tool for assessing and understanding the mechanisms of human MC and developing new therapeutic interventions.
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