Increased Expression of Inflammatory Cytokines in Diseased Cervical Intervertebral Disc is Associated with Discogenic Neck Pain

doi:10.21203/rs.3.rs-2627388/v1

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Increased Expression of Inflammatory Cytokines in Diseased Cervical Intervertebral Disc is Associated with Discogenic Neck Pain

https://doi.org/10.21203/rs.3.rs-2627388/v1

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Background: Chronic neck pain is frequently encountered in clinical practice, but its etiology is still poorly understood. The degenerative cervical intervertebral disc is the main cause of chronic neck pain reported, accounting for about 16%-41% of all incidents. Inflammation is associated with intervertebral disc degeneration and the occurrence of discogenic low back pain; however, whether inflammation plays a key role in the pathogenesis of discogenic neck pain has not been reported in the literature. The aim of the present study was to explore the relationship between inflammatory cytokines and discogenic neck pain and to elucidate the possible pathogenesis of discogenic neck pain.

Methods: A total of 111 intervertebral disc samples were collected during the present study: 38 samples from the discogenic neck pain group, 41 samples from the symptomatic control group, and 32 samples from the normal control group. The concentration in each sample of NO, IL-1, IL-6 and TNF- α was determined using ELISA and then statistically analyzed as to whether there were significant differences among the three groups.

Results: The expression of inflammatory cytokines in disc samples from patients with severe neck pain was increased in comparison with the disc samples from patients without or with only mild neck pain and fresh cadavers. The differences are statistically significant（P＜0.001). However, the difference of the degree of disc degeneration was not statistically significant between the discogenic neck pain group and symptomatic control group（P＞0.05).

Conclusions: The increased expression of inflammatory cytokines in diseased cervical intervertetral discs may play a key role in the pathogenesis of discogenic neck pain. Although inflammation is involved in intervertebral disc degeneration, there is no linear positive correlation between the concentration of inflammatory cytokines and the degree of disc degeneration.

intervertebral disc degeneration

discogenic neck pain

inflammatory cytokines

pathogenesis

discogenic low back pain

Neck pain is frequently encountered in clinical practice. Approximately 66% of adults experience neck pain in their lifetime, and approximately 14% develop chronic neck pain. While it has become a serious economic and social problem, the etiology is still poorly understood [1, 2]. It has been reported that many cervical disorders may cause neck pain, such as neck muscle tension or strain, minor arthropathy, trauma, cervical disc herniation, abnormal spine curvature, and cervical disc degeneration [3]. Although there is no direct evidence that degenerative cervical discs are a source of chronic neck pain, it is reasonable to believe that this is true for some patients based on previous studies and clinical practice. It is often found that neck pain is significantly relieved or even resolved following the resolution of neurological symptoms in patients with cervical spondylotic radiculopathy or myelopathy after undergoing anterior cervical discectomy and fusion (ACDF) or artificial cervical intervertebral disc replacement [4]. In addition, neck pain can be effectively relieved by performing an intervertebral disc block test on patients suffering from chronic neck pain [5]. Therefore, it is reasonable to conclude that degenerated cervical discs are a source of chronic neck pain. Neck pain related to degenerative intervertebral discs can usually be divided into two types [6]: radiation pain or discogenic pain. Radiation pain is caused by spinal stenosis or cervical radiculopathy. In contrast, discogenic pain is caused by disorders of the intervertebral discs and is usually accompanied by neck stiffness, headache, unilateral or bilateral shoulder pain, non-radicular upper arm pain, ocular and vestibular dysfunction, and anterior chest wall pain [7]. It accounts for a large proportion of chronic neck pain, with a reported incidence of 16–41% [2, 5].

Scholars once thought that the intervertebral disc was not innervated; therefore, the diagnosis of discogenic pain was controversial. However, recent studies have shown that intervertebral discs, particularly degenerative ones, have extensive innervation. Fujimoto et al. confirmed in rats that nerve fibers innervating the cervical disc include afferent pain sensory-related nerves and efferent sympathetic and parasympathetic nerves [8]. In 1988, Bogduk et al. described the innervation patterns of the sinuvertebral nerves into the outer third of the annulus fibrosis based on microdissection and histologic studies from clinical patients and cadavers [9]. Their research also demonstrated the presence of nerve fibers and nerve endings within the outer third of the annulus fibrosis using cholinesterase staining. Moreover, additional studies have reported the presence of free nerve endings within human degenerative cervical intervertebral discs [8–11]. Substance P and calcitonin gene-related peptides have also been found in painful lumbar and cervical discs in humans [12, 13]. These studies provide an important anatomical basis for the generation and conduction of discogenic neck pain.

Previous research has shown that inflammation is associated with intervertebral disc degeneration [14, 15]. Moreover, the expression of inflammatory cytokines in degenerative cervical discs has been found to be greater than that of normal discs [10, 16, 17]. As main frontier cytokines, nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) play key roles in the occurrence and progression of intervertebral disc degeneration and discogenic low back pain [18]. However, the possibility of a similar mechanism of pathogensis existing in discogenic neck pain has not been explored in the literature. Therefore, this study aims to determine the expression of inflammatory cytokines in the diseased cervical discs of patients with severe chronic neck pain and to further explore the possible relationship between them.

Samples of cervical discs from cervical spondylotic myelopathy patients with discogenic neck pain were obtained between October 1, 2021, and October 1, 2022. The inclusion criteria was 1) suffering severe chronic neck pain (visual analog scale [VAS] ≥ 70 mm); 2) ≤ 2 disease segments; 3) nonresponse to conservative therapy over a minimum of three months; 4) without a history of cervical trauma, surgery, tuberculosis, infections, tumors, or any demonstrable abnormalities of the congenital or developmental cervical spine. Discogenic neck pain was determined by a positive response to an analgesic discography test [19].

During the analgesic discography test, no contrast medium was used. If two discs were suspected, the second disc was tested after any effects on the first disc had ceased. While in the supine position, under the guidance of a C-armed X-wire machine, a 22-G spinal needle was percutaneously punctured into the center of the disc via an anterolateral approach, as judged from anteroposterior and lateral views. A small volume (0.3–0.5 mL) of 0.25% bupivacaine was injected. If there was no complaint of discomfort after lying supine for two hours, the patient was allowed to get up and move around without limiting cervical spine movement. Pain relief of ≥ 70% was regarded as a positive. Relief percent was calculated using:

\(\left[\right(preoperative VAS – postoperative VAS) / preoperative VAS] \times 100\) ).

Of the 41 patients tested, 28 had a positive response. One patient could not tolerate the surgery due to severe coronary disease; however, the remaining 27 patients underwent anterior cervical surgery using anterior cervical corpectomy and fusion (ACCF) or ACDF surgical procedures, during which 38 cervical disc samples were obtained, as seen in group A. There were 15 men and 12 women in group A, and the mean age was 57.6 years (range: 37–82 years). The mean duration of neck pain was 13.6 months (range: 3–29 months). The involved surgical levels in the current study were from C3/4 to C6/7.

Simultaneously, we collected 41 cervical disc samples from 29 patients who suffered cervical spondylotic myelopathy without neck pain or with only mild neck pain (VAS ≤ 30 mm) and who had undergone ACDF or ACCF as a symptomatic control group. These were labeled as group B, with 14 men and 15 women and a mean age of 55.8 years (range: 33–86 years). The surgical levels involved in the current study were also from C3/4 to C6/7.

In addition, 32 disc samples were collected from eight fresh cadavers as a normal control group, classified as group C. Five men and three women were in this group, with a mean age of 54.3 years (range: 24–65 years). Before collecting samples, the discs showed no signs of degeneration in the imaging examination.

Before the surgery, two experienced radiologists and two senior spine surgeons were invited to evaluate the degeneration degree of the target discs using T2-weighted sagittal magnetic resonance imaging (MRI) (3.0T, Philips) according to the Miyazaki disc degeneration grading system [20]. A detailed record was made, and the procedure was performed under double-blind conditions. The MRI scan (T2W1, TR 2000 ms and TE 90 ms) layer was 4 mm thick, 1 mm apart, and the matrix was 320x192. Discs were scored according to the cervical intervertebral disc degeneration grading system (grades 1–5 of ascending severity), and the score corresponded with the severity grade of the degree of intervertebral disc degeneration. For example, grade 3 corresponds to 3 points, and grade 4 to 4 points. The average of the four scores determined by the two radiologists and two spinologists was taken and regarded as the final disc score.

Written informed consent from each patient was obtained before the research, and the protocol was approved by the Ethics Committee of Tianjing Hospital (2021023).

2.1. Expression of inflammatory cytokines

The 111 disc samples (38, 41, and 32 in groups A, B, and C, respectively) were stored at -80℃. Samples were repeatedly rinsed with normal saline and dried by blotting with absorbent paper in order to accurately weigh each tissue sample. Normal saline was added at a weight-to-volume ratio of 1:4. The discs from each group were then crushed using a Soniprep150 (Sanyo, UK) ultra-medium ultrasonic pulverizer, which produced a 20% tissue homogenate by treating the sample at an amplitude of 14 µm for 30 s. The homogenate was then centrifuged at 3,000 rpm (4°C) for 15 minutes. The supernatant was carefully collected and stored in a refrigerator at 6℃. Assays were completed within 48 hours.

The expression of NO in each sample was determined using nitrate reductase to convert NO to NO₂^- as NO is chemically active and rapidly metabolizes to NO₂^- and NO₃^- in vivo. The result was determined based on the color development depth. We used the standard curve method and strictly adhered to the instructions in the NO test kit offered by Shanghai Xuan Ya Biotechnology Co., Ltd (Shang Hai, China). The amount of NO₂^-/ NO₃^- represented the concentration of NO in the sample expressed as µmol/g protein.

The expression of IL-1, IL-6, and TNF-α was subsequently measured. The samples were thawed at room temperature in accordance with the operating instructions of the ELISA kits (Shanghai Xuan Ya Biotechnology Co., Ltd., Shanghai, China). The concentration of IL-1, IL-6, and TNF-α in each sample was calculated and recorded using a standard curve and expressed in units of pg/g.

2.2. Statistical analysis

Statistical analysis of the data was performed using IBM SPSS Statistics version 20.0 (IBM Corp., Armonk, N.Y., USA). The baseline data of age, sex, and the degree of disc degeneration was tested using one-way analysis of variance, (post hoc, least significance difference [LSD]), Pearson chi-square, and unpaired t-test, respectively. The measurement data (concentration of inflammatory cytokines) were analyzed using one-way analysis of variance (post hoc, LSD) among the three groups. P-value < 0.05 was considered statistically significant.

A total of 111 disc samples were examined in this research. The baseline characteristics of all subjects are shown in Table 1.

Table 1. Baseline characteristics of the 56 patients and eight donors in the three groups
				P-Values
Characteristics	A	B	C	A/B	B/C	A/C

Age ( x̄±s )	57.6±11.92	55.8±13.32	54.3±9.71	0.545	0.810	0.558
Female	12(44.4)	15(51.7)	3(37.5)
Male	15(55.6)	14(48.3)	5(62.5)
Degree of degeneration	4.3±0.63	4.1±0.67	— —
Level of disc
C3/4	2(5.3)	1(2.4)	8(25)
C4/5	12(31.5)	16(39.1)	8(25)
C5/6	15(39.5)	19(46.3)	8(25)
C6/7	9(23.7)	5(12.2)	8(25)

Key: n(%), x±s = mean±standard deviation, A = discogenic neck pain group, B = symptomatic control group, and C = normal control group

There were no statistically significant differences in the demographic or clinical features (one-way analysis of variance, post hoc, LSD; P of age = 0.545, 0.810, and 0.558 between groups A and B, B and C, and A and C, respectively) among the three groups. Significant sex differences were not observed among the three groups (Pearson chi-square, P = 0.733), and no significant differences in the degree of degeneration of the disc samples were found between groups A and B (unpaired t-test; P = 0.176). The frequency of the four segments in group A was similar to that in group B.

According to the ELISA test kit results, concentration of inflammatory cytokines in the normal control group was the lowest among the three groups. Concentration in the symptomatic control group was greater than that of the normal control group, but less than that of discogenic neck pain group. Among the three groups, the concentration of inflammatory cytokines in disc samples from the discogenic neck pain group was the greatest . Differences between the discogenic neck pain group and symptomatic control group, the discogenic neck pain group and normal control group, and the symptomatic control group and normal control group, were statistically significant (P ＜ 0.001 for all). The concentration of inflammatory cytokines is detailed in Table 2.

Table 2. Expression of inflammatory cytokines in the disc samples from groups A, B, and C
	A	B	C	P-Values
				A/B	B/C	A/C
Cytokines
NO	9.89±1.75	7.15±2.78	6.11±1.37	P＜0.001
IL-1β	10.74±1.92	8.03±1.87	5.84±2.25	P＜0.001
IL-6	31.65±2.46	25.79±2.12	20.65±1.26	P＜0.001
TNF-α	5.96±1.91	4.18±2.87	2.05±0.58	P＜0.001

Note. The data show the concentration of inflammatory cytokines in the cervical disc samples from patients in groups A, B, and C. Key: x±s = mean±standard deviation; A = discogenic neck pain group, B = symptomatic control group, C = normal control group.

In addition, we analyzed the original data of the concentration of inflammatory cytokines using Origin Pro 2023 (The Ultimate Software for Graphing and Analysis, OriginLab Corporation). Figure 1 shows the distribution of the data of NO and TNF-α among the three groups. The mean, median, and maximal value of NO are the greatest in group A and lowest in group C. The data distribution of TNF-α among the three groups shows the same trend. Figure 2 shows the same trend in the distribution of the data of IL-1β and IL-6 among the three groups.

Chronic neck pain, similar to chronic low back pain, has become a severe public health problem worldwide [1, 5]. Research in this field has always been the focus of scholars due to the limited understanding of its etiology. As an important component of the physiological structure of the cervical spine, the cervical intervertebral disc has been established as a main source of cervical pain [8, 10]. However, the diagnosis of discogenic neck pain is debatable, as there is no standardized diagnostic method established thus far. Discography, the only test that links the disease with clinical symptoms, is still controversial [2]. This dissension is primarily due to two aspects: 1) false positives are inevitable as increased pressure may induce pain within a normal intervertebral disc [5] and 2) degeneration may be induced or accelerated in normal intervertebral discs due to a puncture injury. Discography is usually only conducted on discs that show degenerative signs on imaging. In order to ensure surgical effects, discography is conducted before surgery; the discs showing normal signs on imaging are not usually referred for discography [4]. Because the discography needle is 22G, the puncture injury is negligible.

In current research, analgesic discography has been adopted, as it is the most effective test for diagnosis and location in painful-disc syndrome [19]. It can not only overcome the disadvantages of conventional discography, such as the high false positive rate resulting from the lack of specific imaging signs and pain style, but also identify the disc responsible for pain and aid in localizing the diagnosis. Although false-negative responses are indeed inevitable as local anesthetic can not fully anesthetize all sources of pain, this may only cause some deviation in the sample size of the discogenic neck pain group and does not affect the results of the current study because patients with negative results have been excluded. Moreover, the suspected discs were injected with only 0.3–0.5 mL bupivacaine. Even if a small amount of bupivacaine leaked out of the cervical intervertebral disc, it could not have reached the posterior facet joints, nor could it have produced an anesthetic effect in the cervical nerve roots. Therefore, any false-positive responses can be effectively excluded.

According to the results of this research, we found that the expression of inflammatory cytokines in cervical disc samples from patients in the discogenic neck pain group increased significantly compared to the symptomatic control group. The two groups have no statistical difference in baseline characteristics; therefore, it is reasonable for us to conclude that the increased expression of inflammatory cytokines may play a key role in the pathomechanism of discogenic neck pain. In addition, no statistical difference in the degree of degeneration has been found between the discogenic neck pain group and the symptomatic control group, which may indicate that there is no linear positive correlation between the concentration of inflammatory cytokines and the degree of disc degeneration. Moreover, the expression of inflammatory cytokines in the diseased cervical discs both from the discogenic neck pain group and symptomatic control group is greater than that in the normal control group. This may additionally confirm the conclusion that inflammation is involved in the process of intervertebral disc degeneration [17, 21–23]. A similar viewpoint can also be found in previous studies that mostly focused on discogenic low back pain. Recently, Peng et al., Wu et al., and Yang et al. reported that the expression of inflammatory cytokines in painful cervical discs was increased [4, 10, 13]. However, because the conclusion was drawn from a comparison with normal disc samples, the interference from the relationship between inflammation and disc degeneration could not be excluded effectively. We compared the expression of inflammatory cytokines in cervical disc samples from the discogenic neck pain group and symptomatic control group because there is no statistical difference in the degree of degeneration between the two groups; therefore, the conclusions drawn from this study are more direct and reliable. Such a comparison has not been reported in previous studies.

Increased expression of inflammatory cytokines, such as NO, TNF-α, IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, and interferon γ, are prominent features of degenerative discs which can promote the degradation of the extracellular matrix, the release of chemokines, and alteration of the cellular phenotype [18]. Interactively, the released chemokines can promote the infiltration and activation of T cells, B cells, and macrophages, further amplifying the inflammatory cascade and promoting the release of neurotrophins, particularly nerve growth factor. Moreover, the inflammatory state within the degenerative disc triggers multiple pathogenic responses, such as cellular senescence and apoptosis, ingrowth of nerves and blood vessels, and discogenic pain [22, 24]. This is consistent with the results of the current study; however, the occurrence of discogenic neck pain is a very complex pathological process, and its exact pathomechanism requires further in-depth study. Whether there is a potential link between the degree of disc degeneration and discogenic pain is still inconclusive because not all degenerative intervertebral discs can cause pain. Furthermore, although we found that the expression of inflammatory cytokines is increased in painful discs, the increased expression of inflammatory cytokines does not always cause pain. The expression of inflammatory cytokines in the cervical disc samples of the symptomatic control group was more than that in the normal control group. However, the patients from this group only suffer from mild neck pain, at most. If there is a threshold of neck pain, the value of this threshold is worth studying.

According to the results of current and previous research, an increased expression of inflammatory cytokines in diseased cervical discs may interact with abnormally ingrowing nerve endings, substance P, and calcitonin gene-related peptides to produce an amplification called ‘peripheral sensitization’ for mechanical stimuli and compressive stress that are below normal thresholds. Eventually this leads to the occurrence of discogenic neck pain [8]. However, due to the complexity of discogenic neck pain, its detailed pathogenesis needs to be further studied.

4.1. Limitations

There are several limitations of the present study. Firstly, we determined the expression of inflammatory cytokines in each sample via ELISA test kits which process the whole intervertebral disc into a tissue homogenate. The results would be more robust if the disc could be tested separately according to the source site, such as the outer layer, inner layer, or nucleus pulposus. Secondly, the inclusion of patients in the symptomatic control group was mainly based on the condition of VAS ≤ 30 mm in this study. It is known that pain is a subjective feeling, and different individuals may tolerate it differently, so whether this factor has biased the results is unclear. In addition, the sample size in this study was small; studies with larger sample sizes are warranted to verify these conclusions.

Based on our current research results, we infer that the increased expression of inflammatory cytokines in diseased cervical intervertebral discs may play a key role in the pathogenesis of discogenic neck pain. Although inflammation is involved in intervertebral disc degeneration, there is no linear positive correlation between the concentration of inflammatory cytokines and the degree of disc degeneration.

ACCF: anterior cervical corpectomy and fusion

ACDF: anterior cervical disectomy and fusion

NO: nitrous oxide

IL-1: interleukin 1

IL-6: interleuin 6

LSD: least significance difference

TNF-α: tumor necrosis factor alpha

Ethics approval and consent to participate: This study was approved by the Ethics Committee of Tianjing Hospital (2021023). Informed consent was obtained from all study participants and the study was carried out in accordance with relevant guidelines and regulations of the Declaration of Helsinki.

Consent for publication: Not applicable.

Availability of data and materials: The datasets used during the current study available from the corresponding author on reasonable request.

Competing interests: The authors declare that there are no competing interests regarding the publication of this article.

Funding: This research received no external funding.

Authors’ contributions: “Conceptualization, X.K. and M.Q.; methodology, D.Z., M.L; software, H.X.; validation, X.K., N.L. and B.X.; formal analysis, M.Q.; investigation, M.L, N.L.; resources, H.X.; data curation, X.K.; writing—original draft preparation, X.K.; writing—review and editing, M.Q.; visualization, D.Z.; supervision, B.X.; project administration, B.X.; funding acquisition, not applicable. All authors have read and agreed to the published version of the manuscript.”

Acknowledgments: The authors would like to thank Yongchao LI, Yongjin LI, and Kaihui Zhang for their help in the preparation and submission of the manuscript. The authors would also like to thank Editage (www.editage.cn) for the English language editing.

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No competing interests reported.

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Increased Expression of Inflammatory Cytokines in Diseased Cervical Intervertebral Disc is Associated with Discogenic Neck Pain

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Abstract

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1. Introduction

2. Materials And Methods

2.1. Expression of inflammatory cytokines

2.2. Statistical analysis

3. Results

4. Discussion

4.1. Limitations

5. Conclusions

Abbreviations

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