SPINE Volume 40, Number 14, pp 1101-1107 ©2015, Wolters Kluwer Health, Inc. All rights reserved.
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes Preliminary Controlled Study Ergul Belge Kurutas, PhD,* Mehmet Senoglu, MD,† Kasim Zafer Yuksel, MD,† Velid Unsal, PhD,* and Idris Altun, MD†
Study Design. Oxidative/nitrosative stress in vertebral endplates of patients with low back pain and Modic changes (MCs) (types I, II, and III) endplate changes on magnetic resonance imaging. Objective. The aim of this study was to assess the levels of oxidative/nitrosative stress biomarkers in patients with MCs. Summary of Background Data. Degenerated discs and endplate abnormalities is postulated as a possible source of low back pain. Oxidative/nitrosative stress plays an important the role in various human diseases. However, the presence of oxidative/ nitrosative stress has not been studied in patients with low back pain and endplate changes on magnetic resonance imaging. Methods. Patients with MCI, II, and III (n = 32) and age- and sexmatched controls subjects (n = 15) were enrolled in this study. Also, 3-nitrotyrosine (3-NT) and nitric oxide levels as nitrosative stress biomarkers were measured with enzyme-linked immunosorbent assay. Also, the activities of catalase (CAT) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA) as oxidative stress biomarkers were determined on spectrophotometer. Results. Oxidative/nitrosative stress was confirmed by the significant elevation in nitric oxide, 3-NT, MDA and decreased of CAT and SOD activities in MCI compared with other MCs and the control group (P < 0.05). The highest CAT and SOD activities were found in patients with MCII compared with the other MCs and the control group. However, the levels of nitric oxide, 3-NT, and MDA showed moderate increase in this group (P < 0.05). In addition, the levels of nitrosative stress biomarkers in patients with MCIII were
From the Departments of *Biochemistry, and †Brain and Nerve Surgery, Faculty of Medicine, Sutcu Imam University, Kahramanmaras, Turkey. Acknowledgment date: January 14, 2014. Revision date: July 23, 2014. Acceptance date: September 14, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). No funds were received in support of this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Ergul Belge Kurutas, PhD, Department of Biochemistry, Faculty of Medicine, Sutcu Imam University, Hastane St, Yoruk Selim mah, 46050, Kahramanmaras, Turkey; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000737 Spine
approximated to the control values (P > 0.05). However, the levels of oxidative stress biomarkers in patients with MCIII were slightly higher than that of the control group (P < 0.05). Conclusion. Our findings indicated that oxidative/nitrosative stress in patients with MCI may be aggravated as a result of oxidant/ antioxidant imbalance and it may cause formation of the lesion in these patients. However, the increased antioxidant activities and MDA, 3-NT levels in patients with MCII and MCIII may be an adaptative response to against oxidative/nitrosative stress. Key words: Modic changes, oxidative/nitrosative stress, malondialdehyde, nitrotyrosine. Level of Evidence: 4 Spine 2015;40:1101–1107
L
ow back pain (LBP) with or without leg pain has been one of the most common causes of disability in adults and is a very important disease for early retirement in industrialized societies.1 Degenerative disc disease is the most frequent problem in patients with LBP.2 Modic changes (MCs) are pathological vertebral endplate and bone marrow changes visible on magnetic resonance image (MRI) and may linked with LBP.3 Three different types of MC have been described; type I change (MCI) lesions, considered to be the earliest and the most active stage in the process of MC evolution, are associated with vascular granulation tissue within the subchondral bone, whereas type II (MCII) lesions reflect fatty replacement of the red bone marrow.4 Type III lesions are observed in vertebral bodies with sclerotic changes.5,6 These are thought to reflect the conversion of MC from one type to another, representing different stages of the same pathological process.6–9 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are molecules or molecular fragments containing 1 or more unpaired electrons in atomic or molecular orbitals, which characterizes free radicals with high reactivity.10 The damage promoted by ROS and RNS is termed oxidative stress and nitrosative stress, respectively. These occur in biological systems when there is dysregulation of the redox balance, caused by deficiency of enzymatic and nonenzymatic antioxidants, and/or an overproduction, or altered spatiotemporal distribution of ROS/RNS. On the basis of the most www.spinejournal.com
1101
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1101
26/06/15 5:49 PM
EPIDEMIOLOGY recent discoveries in this field, oxidative and nitrosative stress might be defined as the disturbance in the balance between ROS/RNS acting as oxidants and levels of protecting antioxidant defense systems in favor of the former, potentially leading to damage and linked to a disruption of redox signaling and control.11 Increased ROS/RNS production is observed in injured peripheral nerve and inflamed tissue and antioxidants produce analgesia in both neuropathic and inflammation pain. However, the underlying mechanism by which ROS reduction alleviates pain is not well understood.12 According to our knowledge, no studies were reported in the literature investigating, the levels of oxidative/nitrosative stress biomarkers in patients with MCs. Therefore, we aimed to evaluate the oxidative stress biomarkers such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and nitrosative stress biomarkers such as nitric oxide (NO), 3-nitrotyrosine (3-NT) in patients with MCs.
MATERIALS AND METHODS Patient Selection For 8 months, from November 2012 to July 2013, total 47 patients with for low back and leg pain at department of brain and nerve surgery in Sutcu Imam University were enrolled in the study. Thirty-two of the 47 patients had severe back leg pain and (+) endplate changes were selected as patients with MCs. The remaining 15 patients had severe back and leg pain, and also (−) endplate changes were selected as the control group. The patients and control individuals who were matched in age and sex with an average age of 51.8 ± 13.0 years (mean ± standard deviation) (range, 30–81 yr). Inclusion was based on fulfillment of all of the following criteria: severe chronic low back and leg pain, defined as low back and leg pain persisting for 3 months, with no response to 3-month conservative treatment and severe interference with lifestyle; age, 30 to 81 years; and MR image of the lumbar spine in the last 6 months. Exclusion criteria were back surgery; spondylolisthesis; uncontrolled depression; LBP related to ankylosing spondylitis, infection, tumor, or fracture; obesity evaluated by a body mass index 30 kg/m2; diabetes; tobacco; and presence of sciatica. In the 47 study patients, discogenic pain was diagnosed by physical examination, pain location, radiography, and MRI. Details of patient and control groups were given in Table 1. Ethics Committee of Sutcu Imam University of Medical Sciences approved the study protocol and each subject signed a detailed informed consent form.
Evaluation of Endplate Evaluation was blinded and performed by 3 observers. If at least 2 observers were in agreement, their classification was used to define the endplate change. Fifteen patients without MC on MR image were selected as the control group (Figure 1A). Endplate abnormalities were divided into those with normal intensity signals on MR image (endplate change). There were 10 patients with a MCI signals (low intensity on T1-weighted spin echo images and high intensity on T2-weighted spin echo images) as shown in Figure 1B; 12 patients with a or MCII 1102
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
signals (high intensity on both T1- and T2-weighted spin echo images) (Figure 1C); and 10 patients with a MCIII signal (low intensity on both T1- and T2-weighted spin echo images) in this study (Figure 1D).
Oxidative/Nitrosative Stress Biomarkers Measurements The blood samples were taken from 32 patients and 15 control individuals. Samples were centrifuged at 3000g for 10 minutes at 4ºC. Plasma was separated and buffy coat was discarded by aspiration. Erythrocytes were washed 4 times with cold physiological saline and stored at −70°C until analysis. The CAT activity in erythrocyte was measured in samples by method applied by Beutler.13 The decomposition of the substrate H2O2 was monitored spectrophotometrically at 240 nm. The activity of CAT was expressed as U/g Hb. The SOD activities in erythrocyte were estimated by the use of the method described by Fridovich.14 SOD activity was expressed as U/g Hb. Lipid peroxidation level in the plasma samples was expressed in MDA. Measurement was based on the method of Ohkawa.15 MDA levels were expressed as nmol/mL. NO level in plasma samples was measured using the Griess reaction.16 NO levels were expressed as μmol/L. Also, 3-NT levels in plasma samples were determined with a “sandwich” enzyme-linked immunosorbent assay kit (Bioxytech Nitrotyrosine-EIA; OxisResearch, Portland, OR) according to the manufacturers’ protocol. Then, 3-NT levels were given as nmol/L.
Statistical Analysis Statistical analysis was carried out using SPSS 17.0 software (SPSS, Inc., Chicago, IL) for Windows statistical software. The conformability of the quantitative data to the normal distribution was examined using Kolmogorov-Smirnov test. As the age and sex were distributed normally, the descriptive statistics were presented as mean ± standard deviation. The Mann-Whitney U test was used to compare mean values between groups. The statistical difference was taken as probability value less than 0.05.
RESULTS Ten patients with MCI (5 females, aged 39–63 yr [mean, 49.0 yr]; 5 males, aged 30–81 yr [mean, 55.2 yr]), 12 patients with MCII (6 females, aged 38–79 yr [mean, 53.6 yr]; 6 males, aged 41–56 yr [mean, 49.5 yr]), and 10 patients with MCIII (6 females, aged 35–65 yr [mean, 52.3 yr]; 4 males, aged 37–69 yr [mean, 50.2 yr]) and also 15 controls (8 females, aged 46–62 yr [mean, 54.5 yr]; 7 males, aged 30–77 yr [mean, 49.0 yr]) comprised the study groups. In all cases, we selected patients after lumbar MRI signal changes were detected and verification of the inclusion and exclusion criteria. There were no significant differences both among Modic subgroups and control groups compared with respect to sex and age (P > 0.05). There were statistically significant differences in the levels of oxidative/nitrosative stress biomarkers among MCs (P < 0.05). Although the activities of antioxidant enzymes (CAT, SOD) in patients with MCI were found lowest in all of the MCs, the levels of MDA, NO, and 3-NT were found highest
www.spinejournal.com
July 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1102
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
TABLE 1. Details of All Patients With Low Back and Leg Pain Case
Sex
Age (yr)
Symptom
Diagnosis
Level
Endplate (MRI)
M. S.
Male
30
Low back pain + left leg pain
Disc degeneration
L2–L3
Modic I
A. A.
Male
79
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic I
G. B.
Female
39
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic I
F. D.
Female
40
Low back pain + left leg pain
Disc degeneration
L4–L5
Modic I
E .A.
Male
35
Low back pain + left leg pain
Disc degeneration
L4–L5
Modic I
A. Y.
Female
63
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic I
F. Y.
Female
52
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic I
A. K.
Female
51
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic I
Y. S.
Male
51
Low back pain + right leg pain
Disc herniation (protrusion)
L4–L5
Modic I
M. O.
Male
81
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Modic I
M. A.
Female
79
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic II
D. M.
Female
38
Low back pain + left leg pain
Disc herniation (bulging)
L4–L5
Modic II
I. K.
Male
50
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic II
A. T.
Female
60
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic II
C. U.
Male
55
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic II
D. P.
Female
54
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic II
G. Y.
Female
47
Low back pain + left leg pain
Disc degeneration
L3–L4
Modic II
A. A.
Female
44
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic II
C. A.
Male
50
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic II
N. A.
Male
41
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic II
E. A.
Male
56
Low back pain + right leg pain
Disc degeneration
L5–S1
Modic II
H. T.
Male
45
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic II
M. N.
Male
42
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic III
H. B.
Female
39
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Modic III
Y. T.
Male
69
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic III
H. G.
Female
65
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic III
A. B.
Female
58
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic III
A. G.
Male
53
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic III
H. K.
Female
56
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Modic III
R. K.
Male
37
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic III
C. Y.
Female
61
Low back pain + left leg pain
Disc herniation (protrusion)
L5–S1
Modic III
K. S.
Female
35
Low back pain + right leg pain
Disc degeneration
L4–L5
Modic III
G. Y.
Female
47
Low back pain + left leg pain
Disc herniation (protrusion)
L5–S1
Control
H. A.
Male
47
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Control
F. A.
Female
57
Low back pain + right leg pain
Disc degeneration
L4–L5
Control
I. K.
Male
30
Low back pain + right leg pain
Disc herniation (protrusion)
L4–L5
Control (Continued )
Spine
www.spinejournal.com
1103
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1103
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
TABLE 1. (Continued ) Case
Sex
Age (yr)
Symptom
Diagnosis
Level
Endplate (MRI)
C. K.
Male
30
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Control
E. E.
Male
35
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Control
T. D.
Female
58
Low back pain + left leg pain
Disc degeneration
L4–L5
Control
Z. C.
Female
62
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Control
A. A.
Male
77
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Control
G. K.
Female
58
Low back pain + right leg pain
Disc herniation (protrusion)
L2–L3
Control
H. P.
Female
46
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Control
M. F.
Male
62
Low back pain + left leg pain
Disc degeneration
L5–S1
Control
F. K.
Female
48
Low back pain + right leg pain
Disc degeneration
L5–S1
Control
Y. P.
Male
62
Low back pain + right leg pain
Disc herniation (protrusion)
L4–L5
Control
E .Y.
Female
60
Low back pain + left leg pain
Disc degeneration
L4–L5
Control
MRI indicates magnetic resonance image.
(P < 0.05). The highest antioxidant enzyme activities were found in patients with MCII (Figure 2A, B). However, the levels of MDA, NO, and 3-NT in this Modic type showed a moderate increase (P < 0.05) (Figure 2C–E). The activities of antioxidant enzymes in MCIII were found lower
than that of MCII. In addition, the levels of MDA, NO, and 3-NT in MCIII were found lowest in all of MCs (P < 0.05) (Figure 2A–E). There were significant differences between MCs and the control group. The activities of antioxidant enzyme in patients
Figure 1. MR images of the control group (A), Modic type I (B), Modic type II (C), and Modic type III (D). MRI indicates magnetic resonance image.
1104
www.spinejournal.com
July 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1104
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
Figure 2. A, The activity of CAT in patients with MCs and control subjects. *P < 0.05. B, The activity of SOD in patients with MCs and control subjects *P < 0.05. C, The level of MDA in patients with MCs and control subjects. *P < 0.05. D, The level of NO in patients with MCs and control subjects. *P < 0.05. E, The level of 3-NT in patients with MCs and control subjects. *P < 0.05. CAT indicates catalase; SOD, superoxide dismutase; MDA, malondialdehyde; NO, nitric oxide; 3-NT, 3- nitrotyrosine; MC, Modic change.
with MCI statistically significantly lower than that of the control group (P < 0.05) (Figure 2A, B). Also, the levels of MDA, NO, and 3-NT showed in this group were higher than that of the control group (P < 0.05) (Figure 2C–E). Antioxidant enzyme activities and MDA levels in patients with MCII and III were found higher than that of the control group (Figure 2A–C). There was no significant difference for NO in patients with MCII and MCIII compared with control group (Figure 2D). However, the levels of 3-NT in patients with MCI and MCII were higher than that of the control group (P < 0.05) as shown in Figure 2E.
DISCUSSION This is the first report to describe clear differences in levels of oxidative/nitrosative stress biomarkers in patients with Modic Spine
I, II, and III class vertebral endplate marrow signal changes on MR image. ROS/RNS are constantly formed in the human body and removed by an antioxidant defense system. An imbalance between ROS/RNS and antioxidant defenses in favor of the former via excessive production of ROS/RNS, loss of antioxidant defenses, or both, has been described as oxidative/nitrosative stress.17,18 In this study, our results showed that oxidative/ nitrosative stress in patients with MCI may be aggravate as a result of oxidant/antioxidant imbalance and it may cause formation of the lesion in these patients. There was no study on oxidative/nitrosative stress in patients with MCs. So, we could not do any comparison. However, many authors have some studies related to inflammation parameters in patients with MCs. Burke et al19 reported that increased levels of www.spinejournal.com
1105
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1105
26/06/15 5:49 PM
EPIDEMIOLOGY interleukin-6 (IL-6), a proinflammatory cytokine, have been detected in the intervertebral disc in patients with chronic LBP with MCI signal changes compared with patients with II signal changes. Ohtori et al20 observed that a significant increase in the number of tumor necrosis factor (TNF)-immunoreactive cells in MCI lesions compared with MCII and MCIII (absence of vertebral endplate signal changes) lesions has been found in the vertebral endplates of patients with chronic LBP. These authors noticed that inflammatory cytokines and nerve ingrowth into vertebral endplates may be a cause of discogenic LBP and that type I changes, representing more active inflammation, seem to be mediated by proinflammatory cytokines, whereas type II and III changes could be more quiescent stages of the process. Rannou et al21 reported that low-grade inflammation indicated by high serum high-sensitivity C-reactive protein (hsCRP) level in patients with chronic LBP could point to MCI signal changes. However, Briggs et al22 noticed that no differences hsCRP level between MCI and MCII due to the low sample size of MCI cases. A recent study has shown that few inflammatory cells are found in these discs and therefore the source of the mediators must be cells from the nucleus pulposus itself.23 It is known that such tissue can produce a range of proinflammatory cytokines. Phillips et al23 reported that nucleus pulpous cells are a source of cytokines and chemokines within the intervertebral disc and that these expression patterns are altered in intervertebral disc pathology. Some authors reported that human nucleus pulposus also produces IL-8, IL-1-ß, and TNF-α have been isolated from homogenates of human disc material.19,24 These studies indicate that the upregulation of proinflammatory mediators within the degenerative disc may be the major origin of LBP. It has long been known that there is a strong relationship between inflammation and oxidative/nitrosative stress.11,25 ROS/RNS generated by inflammatory cells not only help to kill pathogens but also act on the inflammatory cells themselves, altering the intracellular redox balance and functioning as signaling molecules involved with the regulation of inflammatory and immunomodulatory genes. ROS/RNS, leading to signaling cascades that trigger the production of proinflammatory cytokines (TNF-α, IL-8, and PGE2) and chemokines.25 We thought that the increased of proinflammatory cytokines is directly or indirectly related to the modulation of the oxidant/antioxidant redox system, may represent a new susceptibility factor in oxidant-induced disc injury that cause abnormal endplates in patients with LBP. The SODCAT system provides the first defense against oxygen toxicity. SOD catalyzes the dismutation of the superoxide anion radical into water and hydrogen peroxide, which is detoxified by CAT activity. The significant SOD and CAT inhibition in MCI could be attributed to a high production of superoxide anion radicals by the SOD enzyme, which has been reported to inhibit CAT activity in case of excess production.26 Both lipid peroxides and their breakdown products, such as MDA and 4-hydroxy-2-nonenal, can directly or indirectly affect many functions integral to cellular and organ homeostasis. In this study, we found that MDA levels in the MCI were higher than that of the other MCs and control. Increased membrane 1106
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
lipid peroxidation may evoke immune and inflammatory response. We thought that increased MDA levels reflect the increased levels of oxidative stress and decreased antioxidant capacity in patients with MCI. Also, we thought that insufficiency of antioxidant barrier may cause oxidative damage in patients with MCI. And, 3-NT and NO are stable markers of nitrosative stress. Increased formation of 3-NT is a marker of oxidative protein modification. In our study, there was difference between patients with MC and control groups, which may be related to the nitration of proteins. Also, increased NO production in patients with MCI may influence NT concentration and thus increased NT content alone. Accordingly, 3-NT may be a sufficiently sensitive marker to Modic-induced changes in nitrosative stress. The other result of our study, antioxidant enzyme activities (CAT, SOD), MDA, and 3-NT levels in patients with MCII were found as very higher than that of the control group. High SOD and CAT activities may indicate high intracellular superoxide anion radicals in patients with MCII. These results of our study indicated that increased antioxidant activities and MDA, 3-NT in patients with MCII may be an adaptive response to increased generation of ROS/RNS. We thought that not only increases the oxidant tolerance of the cells in patients with MCII, but it also turns up the intracellular antioxidant armament of the cells and ensures efficient protection against a further oxidative/nitrosative stress insult. Therefore, in normal conditions, a low level of cellular stress is useful because it activates and maintains the cellular antioxidant defense. An another result of this study, the levels of nitrosative stress biomarkers in patients with MCIII approximated to the control values. However, oxidative stress exists in patients with MCIII, but not nitrosative stress. It may be relationship between increased degenerative changes and oxidative adaptation in patients with MCIII. It was reported that inflammatory chemicals are released because of impairment of the disc, resulting with activation or destruction of dorsal root ganglion (DRG). Nerve growth factor influences neurons of DRG when there is inflammation.26 DRGs, which innerve disc, are highly sensitive to nerve growth factor and therefore, it leads to sprouting of sensitized neurons into disc and posterior horn of spinal cord. Degeneration of DRG neurons in peripheral nerves, and that DRG mitochondria are particularly affected. DRG mitochondria is vulnerable because they are the origin of ROS/RNS production in the DRG neuron.27 This effect may activate cytokines and overproduced ROS/RNS that may play a central role in patients with LBP. We speculate that ROS/RNS secreted from disc of patients with MCs may injure adjacent DRGs and also induce nerve ingrowth from the injured DRGs into the discs. There were some limitations to this study. We examined a small number of cases in each group including elderly people. It has been reported by some authors that, MCs are associated with increasing age, weight, and male sex.28,29 However, there are age-related increases in oxidative/nitrosative stress. There need to be further studies comparing young age and levels of oxidative/nitrosative stress biomarkers in symptomatic endplates.
www.spinejournal.com
July 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1106
26/06/15 5:49 PM
EPIDEMIOLOGY Unfortunately, we studied oxidative/nitrosative stress biomarkers in the blood of patients with MCs. So, we do not know whether oxidative/nitrosative stress within disc. A source of oxidative/nitrosative stress may be disc. Also, we thought that overproduction of ROS/RNS and proinflammatory mediators in abnormal endplates may be one of cause of LBP; however, further study must be required to resolve pathomechanism of pain originated from endplate.
CONCLUSION The decreased or increased antioxidant capacity in blood samples of patients with MCs supports the increased oxidative/nitrosative stress. Furthermore, oxidative/nitrosative stress may explain the link between endplate changes and inflammation. We speculate that MCs possibly resulted from the inflammatory reaction and overproduction ROS/RNS by the toxic substances from degenerative disc and the local release of ROS/RNS and the sequestration of inflammatory cells, within the disc may be an early and important event in the evolution of MCs.
➢ Key Points Degenerated discs and endplate abnormalities is postulated as a possible source of LBP. Oxidative/nitrosative stress plays an important role in various diseases. However, the presence of oxidative/nitrosative stress has not been studied in patients with LBP and endplate changes on MR image. Therefore, we aimed to evaluate the oxidative stress biomarkers such as MDA, SOD, CAT, and nitrosative stress biomarkers such as NO, 3-NT in patients with MCs.
Acknowledgments The authors thank radiologist Murivet Yuksel, MD, for her assistance in evaluating MR images; neurologist Deniz Tuncel, MD, for her assistance in writing the manuscript; and biostatistician Ali Ozer, MD, for his assistance in analyzing statistically.
References
1. Eser O, Gomleksiz C, Sasani M, et al. Dynamic stabilization in the treatment of degenerative disc disease with Modic changes. Adv Orthop .2013:806267. 2. Rea W, Kapur S, Mutagi H. Intervertebral disc as a source of pain. Contin Educ Anaesth Crit Care Pain 2012;12:279–82. 3. Albert HB, Sorensen JS, Christensen BS, et al. Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. Eur Spine J 2013;22:697–707. 4. Modic MT, Steinberg PM, Ross JS, et al. Degenerative disk disease: assessment of changes in vertebral body marrow with MR imaging. Radiol 1988;166:193–9.
Spine
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
5. Kuisma M, Karppinen J, Haapea M, et al. Modic changes in vertebral endplates: a comparison of MR imaging and multislice CT. Skeletal Radiol 2009;38:141–7. 6. Braithwaite I, White J, Saifuddin A, et al. Vertebral endplate (Modic) changes on lumbar spine MRI: correlation with pain reproduction at lumbar discography. Eur Spine J 1998;7:363–8. 7. Vital JM, Gille O, Pointillart V, et al. Course of Modic 1 six months after lumbar posterior osteosynthesis. Spine 2003;28:715–20. 8. Kuisma M, Karppinen J, Niinimäki J, et al. A three-year follow-up of lumbar spine endplate (Modic) changes. Spine 2006;31:1714–8. 9. Jensen TS, Bendix T, Sorensen JS, et al. Characteristics and natural course of vertebral endplate signal (Modic) changes in the Danish general population. BMC Musculoskelet Disord 2009;10:81. 10. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. New York: Oxford University Press; 1999. 11. Jones DP. Redefining oxidative stress. Antioxid Redox Signal 2006;8:1865–79. 12. Schwartz ES, Kim HY, Wang J, et al. Persistent pain is dependent on spinal mitochondrial antioxidant levels. J Neurosci 2009;29: 159–68. 13. Beutler E. Red Cell Metabolism. A Manual of Biochemical Methods. 2nd ed. New York: Grune and Stratton Inc; 1984:68–70. 14. Fridovich I. Superoxide dismutase. Adv Enzymol 1974;41:35–97. 15. Ohkawa H, Ohishi N, Tagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95: 351–8. 16. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440–3. 17. Nathan C. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J Clin Invest 2003;111:769–78. 18. Kröoncke KD. Nitrosative stress and transcription. Biol Chem 2003;384:1365–77. 19. Burke JG, Watson RW, McCormack D, et al. Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators. J Bone Joint Surg Br 2002;84:196–201. 20. Ohtori S, Inoue G, Ito T, et al. Tumor necrosis factor-immunoreactive cells and PGP 9.5-immunoreactive nerve fibers in vertebral endplates of patients with discogenic low back pain and Modic type 1 or type 2 changes on MRI. Spine 2006;31:1026–31. 21. Rannou F, Ouanes W, Boutron I, et al. High-sensitivity C-reactive protein in chronic low back pain with vertebral endplate Modic signal changes. Arthritis Rheum 2007;57:1311–5. 22. Briggs AM, O’Sullivan PB, Foulner D, et al. Vertebral bone mineral measures and psychological wellbeing among individuals with Modic changes. Clin Med Insights Case Rep 2012;5:35–41. 23. Phillips KL, Chiverton N, Michael AL, et al. The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc. Arthritis Res Ther 2013;15:R213. 24. Takahashi H, Suguro T, Okazime Y, et al. Inflammatory cytokines in the herniated disc of the lumbar spine. Spine 1996;21:218–24. 25. Joshua J, Karina C. Inflammation, Immunity, and Redox Signaling: Inflammation, Chronic Diseases and Cancer-Cell and Molecular Biology, Immunology, and Clinical Bases.Khatami M, ed. Rijeka, Croatia: In Tech; 2012:145–58. Available at: http://www.intechopen.com. 26. Liu Q, Jin L, Shen FH, et al. Fullerol nanoparticles suppress inflammatory response and adipogenesis of vertebral bone marrow stromal cells—a potential novel treatment for intervertebral disc degeneration. Spine J 2013;13:1571–80. 27. Freemont AJ, Watkins A, Maitre CL, et al. Nerve growth factor expression and innervation of the painful intervertebral disc. J Pathol 2002;197:286–92. 28. Karchevsky M, Schweitzer ME, Carrino JA, et al. Reactive endplate marrow changes: a systematic morphologic and epidemiologic evaluation. Skeletal Radiol 2005;34:125–9. 29. Leboeuf-Yde C, Kjaer P, Bendix T, et al. Self-reported hard physical work combined with heavy smoking or overweight may result in so-called Modic changes. BMC Musculoskelet Disord 2008;9:5.
www.spinejournal.com
1107
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1107
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes Preliminary Controlled Study Ergul Belge Kurutas, PhD,* Mehmet Senoglu, MD,† Kasim Zafer Yuksel, MD,† Velid Unsal, PhD,* and Idris Altun, MD†
Study Design. Oxidative/nitrosative stress in vertebral endplates of patients with low back pain and Modic changes (MCs) (types I, II, and III) endplate changes on magnetic resonance imaging. Objective. The aim of this study was to assess the levels of oxidative/nitrosative stress biomarkers in patients with MCs. Summary of Background Data. Degenerated discs and endplate abnormalities is postulated as a possible source of low back pain. Oxidative/nitrosative stress plays an important the role in various human diseases. However, the presence of oxidative/ nitrosative stress has not been studied in patients with low back pain and endplate changes on magnetic resonance imaging. Methods. Patients with MCI, II, and III (n = 32) and age- and sexmatched controls subjects (n = 15) were enrolled in this study. Also, 3-nitrotyrosine (3-NT) and nitric oxide levels as nitrosative stress biomarkers were measured with enzyme-linked immunosorbent assay. Also, the activities of catalase (CAT) and superoxide dismutase (SOD), and the levels of malondialdehyde (MDA) as oxidative stress biomarkers were determined on spectrophotometer. Results. Oxidative/nitrosative stress was confirmed by the significant elevation in nitric oxide, 3-NT, MDA and decreased of CAT and SOD activities in MCI compared with other MCs and the control group (P < 0.05). The highest CAT and SOD activities were found in patients with MCII compared with the other MCs and the control group. However, the levels of nitric oxide, 3-NT, and MDA showed moderate increase in this group (P < 0.05). In addition, the levels of nitrosative stress biomarkers in patients with MCIII were
From the Departments of *Biochemistry, and †Brain and Nerve Surgery, Faculty of Medicine, Sutcu Imam University, Kahramanmaras, Turkey. Acknowledgment date: January 14, 2014. Revision date: July 23, 2014. Acceptance date: September 14, 2014. The manuscript submitted does not contain information about medical device(s)/drug(s). No funds were received in support of this work. No relevant financial activities outside the submitted work. Address correspondence and reprint requests to Ergul Belge Kurutas, PhD, Department of Biochemistry, Faculty of Medicine, Sutcu Imam University, Hastane St, Yoruk Selim mah, 46050, Kahramanmaras, Turkey; E-mail: [email protected] DOI: 10.1097/BRS.0000000000000737 Spine
approximated to the control values (P > 0.05). However, the levels of oxidative stress biomarkers in patients with MCIII were slightly higher than that of the control group (P < 0.05). Conclusion. Our findings indicated that oxidative/nitrosative stress in patients with MCI may be aggravated as a result of oxidant/ antioxidant imbalance and it may cause formation of the lesion in these patients. However, the increased antioxidant activities and MDA, 3-NT levels in patients with MCII and MCIII may be an adaptative response to against oxidative/nitrosative stress. Key words: Modic changes, oxidative/nitrosative stress, malondialdehyde, nitrotyrosine. Level of Evidence: 4 Spine 2015;40:1101–1107
L
ow back pain (LBP) with or without leg pain has been one of the most common causes of disability in adults and is a very important disease for early retirement in industrialized societies.1 Degenerative disc disease is the most frequent problem in patients with LBP.2 Modic changes (MCs) are pathological vertebral endplate and bone marrow changes visible on magnetic resonance image (MRI) and may linked with LBP.3 Three different types of MC have been described; type I change (MCI) lesions, considered to be the earliest and the most active stage in the process of MC evolution, are associated with vascular granulation tissue within the subchondral bone, whereas type II (MCII) lesions reflect fatty replacement of the red bone marrow.4 Type III lesions are observed in vertebral bodies with sclerotic changes.5,6 These are thought to reflect the conversion of MC from one type to another, representing different stages of the same pathological process.6–9 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are molecules or molecular fragments containing 1 or more unpaired electrons in atomic or molecular orbitals, which characterizes free radicals with high reactivity.10 The damage promoted by ROS and RNS is termed oxidative stress and nitrosative stress, respectively. These occur in biological systems when there is dysregulation of the redox balance, caused by deficiency of enzymatic and nonenzymatic antioxidants, and/or an overproduction, or altered spatiotemporal distribution of ROS/RNS. On the basis of the most www.spinejournal.com
1101
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1101
26/06/15 5:49 PM
EPIDEMIOLOGY recent discoveries in this field, oxidative and nitrosative stress might be defined as the disturbance in the balance between ROS/RNS acting as oxidants and levels of protecting antioxidant defense systems in favor of the former, potentially leading to damage and linked to a disruption of redox signaling and control.11 Increased ROS/RNS production is observed in injured peripheral nerve and inflamed tissue and antioxidants produce analgesia in both neuropathic and inflammation pain. However, the underlying mechanism by which ROS reduction alleviates pain is not well understood.12 According to our knowledge, no studies were reported in the literature investigating, the levels of oxidative/nitrosative stress biomarkers in patients with MCs. Therefore, we aimed to evaluate the oxidative stress biomarkers such as malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and nitrosative stress biomarkers such as nitric oxide (NO), 3-nitrotyrosine (3-NT) in patients with MCs.
MATERIALS AND METHODS Patient Selection For 8 months, from November 2012 to July 2013, total 47 patients with for low back and leg pain at department of brain and nerve surgery in Sutcu Imam University were enrolled in the study. Thirty-two of the 47 patients had severe back leg pain and (+) endplate changes were selected as patients with MCs. The remaining 15 patients had severe back and leg pain, and also (−) endplate changes were selected as the control group. The patients and control individuals who were matched in age and sex with an average age of 51.8 ± 13.0 years (mean ± standard deviation) (range, 30–81 yr). Inclusion was based on fulfillment of all of the following criteria: severe chronic low back and leg pain, defined as low back and leg pain persisting for 3 months, with no response to 3-month conservative treatment and severe interference with lifestyle; age, 30 to 81 years; and MR image of the lumbar spine in the last 6 months. Exclusion criteria were back surgery; spondylolisthesis; uncontrolled depression; LBP related to ankylosing spondylitis, infection, tumor, or fracture; obesity evaluated by a body mass index 30 kg/m2; diabetes; tobacco; and presence of sciatica. In the 47 study patients, discogenic pain was diagnosed by physical examination, pain location, radiography, and MRI. Details of patient and control groups were given in Table 1. Ethics Committee of Sutcu Imam University of Medical Sciences approved the study protocol and each subject signed a detailed informed consent form.
Evaluation of Endplate Evaluation was blinded and performed by 3 observers. If at least 2 observers were in agreement, their classification was used to define the endplate change. Fifteen patients without MC on MR image were selected as the control group (Figure 1A). Endplate abnormalities were divided into those with normal intensity signals on MR image (endplate change). There were 10 patients with a MCI signals (low intensity on T1-weighted spin echo images and high intensity on T2-weighted spin echo images) as shown in Figure 1B; 12 patients with a or MCII 1102
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
signals (high intensity on both T1- and T2-weighted spin echo images) (Figure 1C); and 10 patients with a MCIII signal (low intensity on both T1- and T2-weighted spin echo images) in this study (Figure 1D).
Oxidative/Nitrosative Stress Biomarkers Measurements The blood samples were taken from 32 patients and 15 control individuals. Samples were centrifuged at 3000g for 10 minutes at 4ºC. Plasma was separated and buffy coat was discarded by aspiration. Erythrocytes were washed 4 times with cold physiological saline and stored at −70°C until analysis. The CAT activity in erythrocyte was measured in samples by method applied by Beutler.13 The decomposition of the substrate H2O2 was monitored spectrophotometrically at 240 nm. The activity of CAT was expressed as U/g Hb. The SOD activities in erythrocyte were estimated by the use of the method described by Fridovich.14 SOD activity was expressed as U/g Hb. Lipid peroxidation level in the plasma samples was expressed in MDA. Measurement was based on the method of Ohkawa.15 MDA levels were expressed as nmol/mL. NO level in plasma samples was measured using the Griess reaction.16 NO levels were expressed as μmol/L. Also, 3-NT levels in plasma samples were determined with a “sandwich” enzyme-linked immunosorbent assay kit (Bioxytech Nitrotyrosine-EIA; OxisResearch, Portland, OR) according to the manufacturers’ protocol. Then, 3-NT levels were given as nmol/L.
Statistical Analysis Statistical analysis was carried out using SPSS 17.0 software (SPSS, Inc., Chicago, IL) for Windows statistical software. The conformability of the quantitative data to the normal distribution was examined using Kolmogorov-Smirnov test. As the age and sex were distributed normally, the descriptive statistics were presented as mean ± standard deviation. The Mann-Whitney U test was used to compare mean values between groups. The statistical difference was taken as probability value less than 0.05.
RESULTS Ten patients with MCI (5 females, aged 39–63 yr [mean, 49.0 yr]; 5 males, aged 30–81 yr [mean, 55.2 yr]), 12 patients with MCII (6 females, aged 38–79 yr [mean, 53.6 yr]; 6 males, aged 41–56 yr [mean, 49.5 yr]), and 10 patients with MCIII (6 females, aged 35–65 yr [mean, 52.3 yr]; 4 males, aged 37–69 yr [mean, 50.2 yr]) and also 15 controls (8 females, aged 46–62 yr [mean, 54.5 yr]; 7 males, aged 30–77 yr [mean, 49.0 yr]) comprised the study groups. In all cases, we selected patients after lumbar MRI signal changes were detected and verification of the inclusion and exclusion criteria. There were no significant differences both among Modic subgroups and control groups compared with respect to sex and age (P > 0.05). There were statistically significant differences in the levels of oxidative/nitrosative stress biomarkers among MCs (P < 0.05). Although the activities of antioxidant enzymes (CAT, SOD) in patients with MCI were found lowest in all of the MCs, the levels of MDA, NO, and 3-NT were found highest
www.spinejournal.com
July 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1102
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
TABLE 1. Details of All Patients With Low Back and Leg Pain Case
Sex
Age (yr)
Symptom
Diagnosis
Level
Endplate (MRI)
M. S.
Male
30
Low back pain + left leg pain
Disc degeneration
L2–L3
Modic I
A. A.
Male
79
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic I
G. B.
Female
39
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic I
F. D.
Female
40
Low back pain + left leg pain
Disc degeneration
L4–L5
Modic I
E .A.
Male
35
Low back pain + left leg pain
Disc degeneration
L4–L5
Modic I
A. Y.
Female
63
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic I
F. Y.
Female
52
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic I
A. K.
Female
51
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic I
Y. S.
Male
51
Low back pain + right leg pain
Disc herniation (protrusion)
L4–L5
Modic I
M. O.
Male
81
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Modic I
M. A.
Female
79
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic II
D. M.
Female
38
Low back pain + left leg pain
Disc herniation (bulging)
L4–L5
Modic II
I. K.
Male
50
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic II
A. T.
Female
60
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic II
C. U.
Male
55
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic II
D. P.
Female
54
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic II
G. Y.
Female
47
Low back pain + left leg pain
Disc degeneration
L3–L4
Modic II
A. A.
Female
44
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic II
C. A.
Male
50
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic II
N. A.
Male
41
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic II
E. A.
Male
56
Low back pain + right leg pain
Disc degeneration
L5–S1
Modic II
H. T.
Male
45
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic II
M. N.
Male
42
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Modic III
H. B.
Female
39
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Modic III
Y. T.
Male
69
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic III
H. G.
Female
65
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Modic III
A. B.
Female
58
Low back pain + right leg pain
Disc degeneration
L3–L4
Modic III
A. G.
Male
53
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Modic III
H. K.
Female
56
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Modic III
R. K.
Male
37
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Modic III
C. Y.
Female
61
Low back pain + left leg pain
Disc herniation (protrusion)
L5–S1
Modic III
K. S.
Female
35
Low back pain + right leg pain
Disc degeneration
L4–L5
Modic III
G. Y.
Female
47
Low back pain + left leg pain
Disc herniation (protrusion)
L5–S1
Control
H. A.
Male
47
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Control
F. A.
Female
57
Low back pain + right leg pain
Disc degeneration
L4–L5
Control
I. K.
Male
30
Low back pain + right leg pain
Disc herniation (protrusion)
L4–L5
Control (Continued )
Spine
www.spinejournal.com
1103
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1103
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
TABLE 1. (Continued ) Case
Sex
Age (yr)
Symptom
Diagnosis
Level
Endplate (MRI)
C. K.
Male
30
Low back pain + right leg pain
Disc herniation (protrusion)
L5–S1
Control
E. E.
Male
35
Low back pain + left leg pain
Disc herniation (bulging)
L5–S1
Control
T. D.
Female
58
Low back pain + left leg pain
Disc degeneration
L4–L5
Control
Z. C.
Female
62
Low back pain + right leg pain
Disc herniation (bulging)
L4–L5
Control
A. A.
Male
77
Low back pain + right leg pain
Disc herniation (bulging)
L5–S1
Control
G. K.
Female
58
Low back pain + right leg pain
Disc herniation (protrusion)
L2–L3
Control
H. P.
Female
46
Low back pain + left leg pain
Disc herniation (protrusion)
L4–L5
Control
M. F.
Male
62
Low back pain + left leg pain
Disc degeneration
L5–S1
Control
F. K.
Female
48
Low back pain + right leg pain
Disc degeneration
L5–S1
Control
Y. P.
Male
62
Low back pain + right leg pain
Disc herniation (protrusion)
L4–L5
Control
E .Y.
Female
60
Low back pain + left leg pain
Disc degeneration
L4–L5
Control
MRI indicates magnetic resonance image.
(P < 0.05). The highest antioxidant enzyme activities were found in patients with MCII (Figure 2A, B). However, the levels of MDA, NO, and 3-NT in this Modic type showed a moderate increase (P < 0.05) (Figure 2C–E). The activities of antioxidant enzymes in MCIII were found lower
than that of MCII. In addition, the levels of MDA, NO, and 3-NT in MCIII were found lowest in all of MCs (P < 0.05) (Figure 2A–E). There were significant differences between MCs and the control group. The activities of antioxidant enzyme in patients
Figure 1. MR images of the control group (A), Modic type I (B), Modic type II (C), and Modic type III (D). MRI indicates magnetic resonance image.
1104
www.spinejournal.com
July 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1104
26/06/15 5:49 PM
EPIDEMIOLOGY
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
Figure 2. A, The activity of CAT in patients with MCs and control subjects. *P < 0.05. B, The activity of SOD in patients with MCs and control subjects *P < 0.05. C, The level of MDA in patients with MCs and control subjects. *P < 0.05. D, The level of NO in patients with MCs and control subjects. *P < 0.05. E, The level of 3-NT in patients with MCs and control subjects. *P < 0.05. CAT indicates catalase; SOD, superoxide dismutase; MDA, malondialdehyde; NO, nitric oxide; 3-NT, 3- nitrotyrosine; MC, Modic change.
with MCI statistically significantly lower than that of the control group (P < 0.05) (Figure 2A, B). Also, the levels of MDA, NO, and 3-NT showed in this group were higher than that of the control group (P < 0.05) (Figure 2C–E). Antioxidant enzyme activities and MDA levels in patients with MCII and III were found higher than that of the control group (Figure 2A–C). There was no significant difference for NO in patients with MCII and MCIII compared with control group (Figure 2D). However, the levels of 3-NT in patients with MCI and MCII were higher than that of the control group (P < 0.05) as shown in Figure 2E.
DISCUSSION This is the first report to describe clear differences in levels of oxidative/nitrosative stress biomarkers in patients with Modic Spine
I, II, and III class vertebral endplate marrow signal changes on MR image. ROS/RNS are constantly formed in the human body and removed by an antioxidant defense system. An imbalance between ROS/RNS and antioxidant defenses in favor of the former via excessive production of ROS/RNS, loss of antioxidant defenses, or both, has been described as oxidative/nitrosative stress.17,18 In this study, our results showed that oxidative/ nitrosative stress in patients with MCI may be aggravate as a result of oxidant/antioxidant imbalance and it may cause formation of the lesion in these patients. There was no study on oxidative/nitrosative stress in patients with MCs. So, we could not do any comparison. However, many authors have some studies related to inflammation parameters in patients with MCs. Burke et al19 reported that increased levels of www.spinejournal.com
1105
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1105
26/06/15 5:49 PM
EPIDEMIOLOGY interleukin-6 (IL-6), a proinflammatory cytokine, have been detected in the intervertebral disc in patients with chronic LBP with MCI signal changes compared with patients with II signal changes. Ohtori et al20 observed that a significant increase in the number of tumor necrosis factor (TNF)-immunoreactive cells in MCI lesions compared with MCII and MCIII (absence of vertebral endplate signal changes) lesions has been found in the vertebral endplates of patients with chronic LBP. These authors noticed that inflammatory cytokines and nerve ingrowth into vertebral endplates may be a cause of discogenic LBP and that type I changes, representing more active inflammation, seem to be mediated by proinflammatory cytokines, whereas type II and III changes could be more quiescent stages of the process. Rannou et al21 reported that low-grade inflammation indicated by high serum high-sensitivity C-reactive protein (hsCRP) level in patients with chronic LBP could point to MCI signal changes. However, Briggs et al22 noticed that no differences hsCRP level between MCI and MCII due to the low sample size of MCI cases. A recent study has shown that few inflammatory cells are found in these discs and therefore the source of the mediators must be cells from the nucleus pulposus itself.23 It is known that such tissue can produce a range of proinflammatory cytokines. Phillips et al23 reported that nucleus pulpous cells are a source of cytokines and chemokines within the intervertebral disc and that these expression patterns are altered in intervertebral disc pathology. Some authors reported that human nucleus pulposus also produces IL-8, IL-1-ß, and TNF-α have been isolated from homogenates of human disc material.19,24 These studies indicate that the upregulation of proinflammatory mediators within the degenerative disc may be the major origin of LBP. It has long been known that there is a strong relationship between inflammation and oxidative/nitrosative stress.11,25 ROS/RNS generated by inflammatory cells not only help to kill pathogens but also act on the inflammatory cells themselves, altering the intracellular redox balance and functioning as signaling molecules involved with the regulation of inflammatory and immunomodulatory genes. ROS/RNS, leading to signaling cascades that trigger the production of proinflammatory cytokines (TNF-α, IL-8, and PGE2) and chemokines.25 We thought that the increased of proinflammatory cytokines is directly or indirectly related to the modulation of the oxidant/antioxidant redox system, may represent a new susceptibility factor in oxidant-induced disc injury that cause abnormal endplates in patients with LBP. The SODCAT system provides the first defense against oxygen toxicity. SOD catalyzes the dismutation of the superoxide anion radical into water and hydrogen peroxide, which is detoxified by CAT activity. The significant SOD and CAT inhibition in MCI could be attributed to a high production of superoxide anion radicals by the SOD enzyme, which has been reported to inhibit CAT activity in case of excess production.26 Both lipid peroxides and their breakdown products, such as MDA and 4-hydroxy-2-nonenal, can directly or indirectly affect many functions integral to cellular and organ homeostasis. In this study, we found that MDA levels in the MCI were higher than that of the other MCs and control. Increased membrane 1106
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
lipid peroxidation may evoke immune and inflammatory response. We thought that increased MDA levels reflect the increased levels of oxidative stress and decreased antioxidant capacity in patients with MCI. Also, we thought that insufficiency of antioxidant barrier may cause oxidative damage in patients with MCI. And, 3-NT and NO are stable markers of nitrosative stress. Increased formation of 3-NT is a marker of oxidative protein modification. In our study, there was difference between patients with MC and control groups, which may be related to the nitration of proteins. Also, increased NO production in patients with MCI may influence NT concentration and thus increased NT content alone. Accordingly, 3-NT may be a sufficiently sensitive marker to Modic-induced changes in nitrosative stress. The other result of our study, antioxidant enzyme activities (CAT, SOD), MDA, and 3-NT levels in patients with MCII were found as very higher than that of the control group. High SOD and CAT activities may indicate high intracellular superoxide anion radicals in patients with MCII. These results of our study indicated that increased antioxidant activities and MDA, 3-NT in patients with MCII may be an adaptive response to increased generation of ROS/RNS. We thought that not only increases the oxidant tolerance of the cells in patients with MCII, but it also turns up the intracellular antioxidant armament of the cells and ensures efficient protection against a further oxidative/nitrosative stress insult. Therefore, in normal conditions, a low level of cellular stress is useful because it activates and maintains the cellular antioxidant defense. An another result of this study, the levels of nitrosative stress biomarkers in patients with MCIII approximated to the control values. However, oxidative stress exists in patients with MCIII, but not nitrosative stress. It may be relationship between increased degenerative changes and oxidative adaptation in patients with MCIII. It was reported that inflammatory chemicals are released because of impairment of the disc, resulting with activation or destruction of dorsal root ganglion (DRG). Nerve growth factor influences neurons of DRG when there is inflammation.26 DRGs, which innerve disc, are highly sensitive to nerve growth factor and therefore, it leads to sprouting of sensitized neurons into disc and posterior horn of spinal cord. Degeneration of DRG neurons in peripheral nerves, and that DRG mitochondria are particularly affected. DRG mitochondria is vulnerable because they are the origin of ROS/RNS production in the DRG neuron.27 This effect may activate cytokines and overproduced ROS/RNS that may play a central role in patients with LBP. We speculate that ROS/RNS secreted from disc of patients with MCs may injure adjacent DRGs and also induce nerve ingrowth from the injured DRGs into the discs. There were some limitations to this study. We examined a small number of cases in each group including elderly people. It has been reported by some authors that, MCs are associated with increasing age, weight, and male sex.28,29 However, there are age-related increases in oxidative/nitrosative stress. There need to be further studies comparing young age and levels of oxidative/nitrosative stress biomarkers in symptomatic endplates.
www.spinejournal.com
July 2015
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1106
26/06/15 5:49 PM
EPIDEMIOLOGY Unfortunately, we studied oxidative/nitrosative stress biomarkers in the blood of patients with MCs. So, we do not know whether oxidative/nitrosative stress within disc. A source of oxidative/nitrosative stress may be disc. Also, we thought that overproduction of ROS/RNS and proinflammatory mediators in abnormal endplates may be one of cause of LBP; however, further study must be required to resolve pathomechanism of pain originated from endplate.
CONCLUSION The decreased or increased antioxidant capacity in blood samples of patients with MCs supports the increased oxidative/nitrosative stress. Furthermore, oxidative/nitrosative stress may explain the link between endplate changes and inflammation. We speculate that MCs possibly resulted from the inflammatory reaction and overproduction ROS/RNS by the toxic substances from degenerative disc and the local release of ROS/RNS and the sequestration of inflammatory cells, within the disc may be an early and important event in the evolution of MCs.
➢ Key Points Degenerated discs and endplate abnormalities is postulated as a possible source of LBP. Oxidative/nitrosative stress plays an important role in various diseases. However, the presence of oxidative/nitrosative stress has not been studied in patients with LBP and endplate changes on MR image. Therefore, we aimed to evaluate the oxidative stress biomarkers such as MDA, SOD, CAT, and nitrosative stress biomarkers such as NO, 3-NT in patients with MCs.
Acknowledgments The authors thank radiologist Murivet Yuksel, MD, for her assistance in evaluating MR images; neurologist Deniz Tuncel, MD, for her assistance in writing the manuscript; and biostatistician Ali Ozer, MD, for his assistance in analyzing statistically.
References
1. Eser O, Gomleksiz C, Sasani M, et al. Dynamic stabilization in the treatment of degenerative disc disease with Modic changes. Adv Orthop .2013:806267. 2. Rea W, Kapur S, Mutagi H. Intervertebral disc as a source of pain. Contin Educ Anaesth Crit Care Pain 2012;12:279–82. 3. Albert HB, Sorensen JS, Christensen BS, et al. Antibiotic treatment in patients with chronic low back pain and vertebral bone edema (Modic type 1 changes): a double-blind randomized clinical controlled trial of efficacy. Eur Spine J 2013;22:697–707. 4. Modic MT, Steinberg PM, Ross JS, et al. Degenerative disk disease: assessment of changes in vertebral body marrow with MR imaging. Radiol 1988;166:193–9.
Spine
Oxidative/Nitrosative Stress in Patients With Modic Changes • Kurutas et al
5. Kuisma M, Karppinen J, Haapea M, et al. Modic changes in vertebral endplates: a comparison of MR imaging and multislice CT. Skeletal Radiol 2009;38:141–7. 6. Braithwaite I, White J, Saifuddin A, et al. Vertebral endplate (Modic) changes on lumbar spine MRI: correlation with pain reproduction at lumbar discography. Eur Spine J 1998;7:363–8. 7. Vital JM, Gille O, Pointillart V, et al. Course of Modic 1 six months after lumbar posterior osteosynthesis. Spine 2003;28:715–20. 8. Kuisma M, Karppinen J, Niinimäki J, et al. A three-year follow-up of lumbar spine endplate (Modic) changes. Spine 2006;31:1714–8. 9. Jensen TS, Bendix T, Sorensen JS, et al. Characteristics and natural course of vertebral endplate signal (Modic) changes in the Danish general population. BMC Musculoskelet Disord 2009;10:81. 10. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. New York: Oxford University Press; 1999. 11. Jones DP. Redefining oxidative stress. Antioxid Redox Signal 2006;8:1865–79. 12. Schwartz ES, Kim HY, Wang J, et al. Persistent pain is dependent on spinal mitochondrial antioxidant levels. J Neurosci 2009;29: 159–68. 13. Beutler E. Red Cell Metabolism. A Manual of Biochemical Methods. 2nd ed. New York: Grune and Stratton Inc; 1984:68–70. 14. Fridovich I. Superoxide dismutase. Adv Enzymol 1974;41:35–97. 15. Ohkawa H, Ohishi N, Tagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95: 351–8. 16. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36:1440–3. 17. Nathan C. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling. J Clin Invest 2003;111:769–78. 18. Kröoncke KD. Nitrosative stress and transcription. Biol Chem 2003;384:1365–77. 19. Burke JG, Watson RW, McCormack D, et al. Intervertebral discs which cause low back pain secrete high levels of proinflammatory mediators. J Bone Joint Surg Br 2002;84:196–201. 20. Ohtori S, Inoue G, Ito T, et al. Tumor necrosis factor-immunoreactive cells and PGP 9.5-immunoreactive nerve fibers in vertebral endplates of patients with discogenic low back pain and Modic type 1 or type 2 changes on MRI. Spine 2006;31:1026–31. 21. Rannou F, Ouanes W, Boutron I, et al. High-sensitivity C-reactive protein in chronic low back pain with vertebral endplate Modic signal changes. Arthritis Rheum 2007;57:1311–5. 22. Briggs AM, O’Sullivan PB, Foulner D, et al. Vertebral bone mineral measures and psychological wellbeing among individuals with Modic changes. Clin Med Insights Case Rep 2012;5:35–41. 23. Phillips KL, Chiverton N, Michael AL, et al. The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc. Arthritis Res Ther 2013;15:R213. 24. Takahashi H, Suguro T, Okazime Y, et al. Inflammatory cytokines in the herniated disc of the lumbar spine. Spine 1996;21:218–24. 25. Joshua J, Karina C. Inflammation, Immunity, and Redox Signaling: Inflammation, Chronic Diseases and Cancer-Cell and Molecular Biology, Immunology, and Clinical Bases.Khatami M, ed. Rijeka, Croatia: In Tech; 2012:145–58. Available at: http://www.intechopen.com. 26. Liu Q, Jin L, Shen FH, et al. Fullerol nanoparticles suppress inflammatory response and adipogenesis of vertebral bone marrow stromal cells—a potential novel treatment for intervertebral disc degeneration. Spine J 2013;13:1571–80. 27. Freemont AJ, Watkins A, Maitre CL, et al. Nerve growth factor expression and innervation of the painful intervertebral disc. J Pathol 2002;197:286–92. 28. Karchevsky M, Schweitzer ME, Carrino JA, et al. Reactive endplate marrow changes: a systematic morphologic and epidemiologic evaluation. Skeletal Radiol 2005;34:125–9. 29. Leboeuf-Yde C, Kjaer P, Bendix T, et al. Self-reported hard physical work combined with heavy smoking or overweight may result in so-called Modic changes. BMC Musculoskelet Disord 2008;9:5.
www.spinejournal.com
1107
Copyright © 2015 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. SPINE140097_LR 1107
26/06/15 5:49 PM
