Original Paper Received: September 16, 2013 Accepted: May 7, 2014 Published online: September 30, 2014
Eur Neurol 2014;72:249–254 DOI: 10.1159/000363515
Oxidative Stress Induced by Lipid Peroxidation Is Related with Inflammation of Demyelination and Neurodegeneration in Multiple Sclerosis Pin Wang a Keqin Xie b Cunfu Wang a Jianzhong Bi a
a Department of Neurology, The Affiliated Hospital of Shandong University and b Institute of Toxicology, Shandong University, Jinan, Shandong, PR China
Key Words RRMS · Methylprednisolone · Oxidative stress · ROS · TAC · MDA
Abstract Background: Multiple sclerosis (MS) is a chronic autoimmune demyelination disorder of the central nervous system (CNS) and its etiology remains unknown. The inflammatory environment in demyelinating lesions leads to the generation of oxygen- and nitrogen-free radicals as well as proinflammatory cytokines, which contribute to the development and progression of multiple sclerosis. Inflammation can lead to oxidative stress and vice versa. Thus, oxidative stress is involved in the inflammation leading demyelination and neurodegeneration in the pathogenesis of multiple sclerosis. Summary: The present study aims to determine two biochemical markers of oxidative stress: TAC and MDA and to show their correlations whether oxidative stress reaction occurs in the demyelination through analyzing samples including peripheral blood and cerebrospinal fluid (CSF) from patients with relapsing-remitting MS (RR-MS). Totally, there were 20 patients in the control groups made from individuals with normal pressure hydrocephalus. Thirty MS patients diagnosed with McDonald diagnostic criteria (2010) treated with methylprednisolone were included in this study. Data were stratified by the degree of severity in order to clarify the role of oxidative stress in the mechanisms of MS and to assess its potential as a biomarker. Thirty clinically definite
© 2014 S. Karger AG, Basel 0014–3022/14/0724–0249$39.50/0 E-Mail [email protected] www.karger.com/ene
RRMS patients were enrolled in this study. Levels of MDA, GSH, total antioxidant capacity TAC, GSH-Px and ROS, were determined in serum of the control group and RRMS patients in 7 days before MP (methylprednisolone) treatment and one month after MP treatment. Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by LSD’s post hoc tests. Key Messages: Oxidative stress precedes the inflammatory response in the multiple sclerosis patients. And methylprednisolone treatment can decrease brain antioxidant enzymes to reduce the neuroinflammato© 2014 S. Karger AG, Basel ry attack.
Introduction
Multiple sclerosis (MS), the most common cause of nontraumatic disability in young adults, is a chronic complex neurological disease with a variable clinical course and several pathophysiological mechanisms such as inflammation, demyelination, axonal/neuronal damage, gliosis, oligodendrocyte loss, remyelination and repair mechanisms, and oxidative stress [1]. Alterations of the immune system together with biochemical disturbances and disruption of the blood-brain barrier are also involved in the pathomechanisms [2]. These processes are not uniformly represented in patient populations but can selectively predominate in individual patients. Therefore, heterogeneity in phenotypic Keqin Xie Institute of Toxicology, Shandong University 44 West Wenhua Road Jinan, Shandong 250012 (PR China) E-Mail keqinx @ sdu.edu.cn
Downloaded by: UCONN Storrs 137.99.31.134 - 5/20/2015 6:12:17 AM
250
Eur Neurol 2014;72:249–254 DOI: 10.1159/000363515
The present study sought to evaluate the oxidative stress in peripheral blood samples and CSF samples from patients with relapsing-remitting MS (RR-MS). Oxidative stress is characterized by an imbalance between ROS/ RNS and antioxidant systems. Data were stratified by the degree of severity in order to clarify the role of oxidative stress in MS and to assess its potential as a biomarker.
Material and Methods Ethics Statement The study was approved by the local Ethics Committee (CEIC Hospital La Paz) and written informed consent was obtained from all subjects. The study complies with the guidelines of the Declaration of Helsinki. Patients Thirty clinically definite RRMS patients were newly diagnosed according to McDonald diagnostic criteria [14]. They were enrolled in this study. The relapsing remitting course of multiple sclerosis patients was ascertained according to Lublin et al. [15]. The clinical diagnosis of RRMS patients, admitted to the Department of Neurology in Shandong University, were based on clinical signs, routine diagnostic CSF and blood tests, full neurological examination and MRI of the central nervous system. The control groups were made from individuals with normal pressure hydrocephalus. The number is 20. The neurologic deficits were scored with the Kurtzke expanded disability status scale (EDSS) [16]. Methylprednisolone (MP) dose of treatment is: 0.4 mg/kg × 5 days, then 30 mg prednisone tablets taken orally once a day. We reduced the dose gradually. Biochemical Investigation Blood samples, with and without EDTA, were collected in the early morning after an overnight fast, within 7 days of an acute exacerbation and one month after MP therapy, twice from RRMS patients and once from controls. Serum and plasma samples were stored at –70 ° C until analysis. CSF samples were taken twice. First time was within 7 days of an acute exacerbation, before MP therapy from RRMS patients, and the second time was one month after MP therapy. The control groups were made from individuals with normal pressure hydrocephalus. CSF samples were collected by lumbar puncture in the L3/L4 or L4/L5 interspace. The first 12 ml of CSF was collected in a polypropylene tube, immediately centrifuged at 2,000 g, at +4 ° C for 10 min. The supernatant was pipetted off, gently mixed to avoid possible gradient effects and aliquoted in polypropylene tubes. These tubes were stored at –80 ° C, waiting for biochemical analysis, without being thawed and refrozen.
Determination of the Lipid Peroxidation and Antioxidation System Lipid Peroxidation (LPO) was determined by measuring the accumulation of thiobarbituric acid-reactive substance in homogenates and expressed as MDA content. The contents of MDA, GSH and the activities of GR, GSH-Px, and TAC (total anti-oxidant capacity) were measured (UV/visible-120–2 Spectrophotometer,
Wang/Xie/Wang/Bi
Downloaded by: UCONN Storrs 137.99.31.134 - 5/20/2015 6:12:17 AM
expression of MS has potential effects on the prognosis and response to therapies in MS patients. Relapsing remitting multiple sclerosis (RRMS) is characterized by periods of clinical stability, punctuated by subacute attacks of clinical worsening after complete or partial recovery [3]. A number of studies report that reactive oxygen species (ROS) play a key role in myelin phagocytosis, contributing to several of the processes underlying MS pathogenesis [4–6]. The inflammatory response gives rise to the production of both ROS and reactive nitrogen species (RNS); ROS production, attributed to monocyte interactions with brain endothelium, induces cytoskeletal rearrangements, loss of blood-brain barrier integrity, tight-junction alterations and the extravasation of leukocytes into the CNS [7, 8]. A number of mechanisms are thought to be involved in the demyelination and neurodegeneration characteristic of MS. Recently, oxidative damage has been associated with mitochondrial injury and energy failure, which may account for certain pathological features of multiple sclerosis, including demyelination, oligodendrocyte apoptosis and astrocyte dysfunction [9]. In RRMS, activated RNS, which can cause nitrosative stress, may lead to myelin destruction. Due to the elevated expression of NOS in activated microglia and infiltrated macrophages, nitric oxide (NO) production is significantly increased in the inflammatory region. Evidence suggests that NO contributes to tissue injury, blood-brain barrier (BBB) breakdown, axonal degeneration and plaque formation. On the other hand, NO may help to control the immune response via several immunoregulatory processes. It is a marker of nitrosative stress in cells and tissues and also a relatively specific marker of oxidative damage, mediated by peroxynitrite, which is formed by the reaction of superoxide with NO [10, 11]. The central nervous system (CNS) is particularly susceptible to oxidative stress due to the high rate of oxygen utilization, a relatively poor antioxidant defence system [12]. In recent years, the role of methylprednisolone (MP) in the RRMS treatment has gained strength. Due to their strong anti-inflammatory action, they are used most effectively to overcome and shorten the duration of neurological symptoms during relapses. MP activity consists mainly of the induction of specific inflammation suppressing genes and repression of genes coding proteins involved in immune and inflammatory reactions. MP therapy results in inhibition of inflammatory reactions, sealing of BBB and decreasing the magnetic resonance imaging (MRI) number of gadolinium up-taking foci [13].
Blood Detection The following items were detected with biochemical methods. Cholesterol (mg/dl), Triglycerides (mg/dl), HDL (mg/dl), LDL (mg/dl), CRP (mg/l), ESR (mm/h). Erythrocytes, WBC (103/μl), Neurophil (103/μl), Basophil (%), Eosinophil (%), Lymphocytes (%), Uric acid (mg/dl), Albumin (mg/dl). The MPO (myeloperoxidase) was determined with MPO Elisa kits. Statistical Analysis The data were expressed as mean ± SD, statistical analysis was performed with one-way analysis of variance (ANOVA), followed by LSD’s post hoc test. A p value of less than 0.05 (* p < 0.05) was considered statistically significant. The statistical analysis was performed using the SPSS v. 17 statistical software packages (SPSS Inc., Chicago, Ill., USA).
Results
Demographic data for study groups are shown in table 1. In addition, basic metabolic profile/blood chemistries did not show significant differences (table 1). RR-MS patients displayed a nonsignificant reduction in uric acid levels (table 1). But in CRP (It is a biomarker of systemic inflammation), there is a minor difference between control and RRMS patients. The value of control is 35.4 ± 3.4 mg/l, while in RRMS patients is 8.4 ± 3.03 mg/l. The value of CRP in MS patients is much lower than that of the control patients. MPO (myeloperoxidase) plays an important role in neutrophil microbicidal action through catalyzing chlorideion oxidation to hypochlorous acid, which is a potent antimicrobial agent. MPO possesses potent pro inflammatory properties and may contribute directly to tissue injury. In addition, MPO is shown to be involved in pathogenesis of lung cancer, Alzheimer’s disease and multiple sclerosis [17]. In our cases, the level of MPO increased significantly in RRMS patients by 120% (** p < 0.01).
Table 1. Subjects’ characteristics and blood chemistries in relaps-
ing-remitting multiple sclerosis (RR-MS) Control group (n = 20) Gender (male/female) Age, years Disease duration, years Scale of EDSS (subjects, n)
Eur Neurol 2014;72:249–254 DOI: 10.1159/000363515
Oxidative Stress Induced by Lipid Peroxidation Is Related with Inflammation of Demyelination and Neurodegeneration in Multiple Sclerosis Pin Wang a Keqin Xie b Cunfu Wang a Jianzhong Bi a
a Department of Neurology, The Affiliated Hospital of Shandong University and b Institute of Toxicology, Shandong University, Jinan, Shandong, PR China
Key Words RRMS · Methylprednisolone · Oxidative stress · ROS · TAC · MDA
Abstract Background: Multiple sclerosis (MS) is a chronic autoimmune demyelination disorder of the central nervous system (CNS) and its etiology remains unknown. The inflammatory environment in demyelinating lesions leads to the generation of oxygen- and nitrogen-free radicals as well as proinflammatory cytokines, which contribute to the development and progression of multiple sclerosis. Inflammation can lead to oxidative stress and vice versa. Thus, oxidative stress is involved in the inflammation leading demyelination and neurodegeneration in the pathogenesis of multiple sclerosis. Summary: The present study aims to determine two biochemical markers of oxidative stress: TAC and MDA and to show their correlations whether oxidative stress reaction occurs in the demyelination through analyzing samples including peripheral blood and cerebrospinal fluid (CSF) from patients with relapsing-remitting MS (RR-MS). Totally, there were 20 patients in the control groups made from individuals with normal pressure hydrocephalus. Thirty MS patients diagnosed with McDonald diagnostic criteria (2010) treated with methylprednisolone were included in this study. Data were stratified by the degree of severity in order to clarify the role of oxidative stress in the mechanisms of MS and to assess its potential as a biomarker. Thirty clinically definite
© 2014 S. Karger AG, Basel 0014–3022/14/0724–0249$39.50/0 E-Mail [email protected] www.karger.com/ene
RRMS patients were enrolled in this study. Levels of MDA, GSH, total antioxidant capacity TAC, GSH-Px and ROS, were determined in serum of the control group and RRMS patients in 7 days before MP (methylprednisolone) treatment and one month after MP treatment. Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by LSD’s post hoc tests. Key Messages: Oxidative stress precedes the inflammatory response in the multiple sclerosis patients. And methylprednisolone treatment can decrease brain antioxidant enzymes to reduce the neuroinflammato© 2014 S. Karger AG, Basel ry attack.
Introduction
Multiple sclerosis (MS), the most common cause of nontraumatic disability in young adults, is a chronic complex neurological disease with a variable clinical course and several pathophysiological mechanisms such as inflammation, demyelination, axonal/neuronal damage, gliosis, oligodendrocyte loss, remyelination and repair mechanisms, and oxidative stress [1]. Alterations of the immune system together with biochemical disturbances and disruption of the blood-brain barrier are also involved in the pathomechanisms [2]. These processes are not uniformly represented in patient populations but can selectively predominate in individual patients. Therefore, heterogeneity in phenotypic Keqin Xie Institute of Toxicology, Shandong University 44 West Wenhua Road Jinan, Shandong 250012 (PR China) E-Mail keqinx @ sdu.edu.cn
Downloaded by: UCONN Storrs 137.99.31.134 - 5/20/2015 6:12:17 AM
250
Eur Neurol 2014;72:249–254 DOI: 10.1159/000363515
The present study sought to evaluate the oxidative stress in peripheral blood samples and CSF samples from patients with relapsing-remitting MS (RR-MS). Oxidative stress is characterized by an imbalance between ROS/ RNS and antioxidant systems. Data were stratified by the degree of severity in order to clarify the role of oxidative stress in MS and to assess its potential as a biomarker.
Material and Methods Ethics Statement The study was approved by the local Ethics Committee (CEIC Hospital La Paz) and written informed consent was obtained from all subjects. The study complies with the guidelines of the Declaration of Helsinki. Patients Thirty clinically definite RRMS patients were newly diagnosed according to McDonald diagnostic criteria [14]. They were enrolled in this study. The relapsing remitting course of multiple sclerosis patients was ascertained according to Lublin et al. [15]. The clinical diagnosis of RRMS patients, admitted to the Department of Neurology in Shandong University, were based on clinical signs, routine diagnostic CSF and blood tests, full neurological examination and MRI of the central nervous system. The control groups were made from individuals with normal pressure hydrocephalus. The number is 20. The neurologic deficits were scored with the Kurtzke expanded disability status scale (EDSS) [16]. Methylprednisolone (MP) dose of treatment is: 0.4 mg/kg × 5 days, then 30 mg prednisone tablets taken orally once a day. We reduced the dose gradually. Biochemical Investigation Blood samples, with and without EDTA, were collected in the early morning after an overnight fast, within 7 days of an acute exacerbation and one month after MP therapy, twice from RRMS patients and once from controls. Serum and plasma samples were stored at –70 ° C until analysis. CSF samples were taken twice. First time was within 7 days of an acute exacerbation, before MP therapy from RRMS patients, and the second time was one month after MP therapy. The control groups were made from individuals with normal pressure hydrocephalus. CSF samples were collected by lumbar puncture in the L3/L4 or L4/L5 interspace. The first 12 ml of CSF was collected in a polypropylene tube, immediately centrifuged at 2,000 g, at +4 ° C for 10 min. The supernatant was pipetted off, gently mixed to avoid possible gradient effects and aliquoted in polypropylene tubes. These tubes were stored at –80 ° C, waiting for biochemical analysis, without being thawed and refrozen.
Determination of the Lipid Peroxidation and Antioxidation System Lipid Peroxidation (LPO) was determined by measuring the accumulation of thiobarbituric acid-reactive substance in homogenates and expressed as MDA content. The contents of MDA, GSH and the activities of GR, GSH-Px, and TAC (total anti-oxidant capacity) were measured (UV/visible-120–2 Spectrophotometer,
Wang/Xie/Wang/Bi
Downloaded by: UCONN Storrs 137.99.31.134 - 5/20/2015 6:12:17 AM
expression of MS has potential effects on the prognosis and response to therapies in MS patients. Relapsing remitting multiple sclerosis (RRMS) is characterized by periods of clinical stability, punctuated by subacute attacks of clinical worsening after complete or partial recovery [3]. A number of studies report that reactive oxygen species (ROS) play a key role in myelin phagocytosis, contributing to several of the processes underlying MS pathogenesis [4–6]. The inflammatory response gives rise to the production of both ROS and reactive nitrogen species (RNS); ROS production, attributed to monocyte interactions with brain endothelium, induces cytoskeletal rearrangements, loss of blood-brain barrier integrity, tight-junction alterations and the extravasation of leukocytes into the CNS [7, 8]. A number of mechanisms are thought to be involved in the demyelination and neurodegeneration characteristic of MS. Recently, oxidative damage has been associated with mitochondrial injury and energy failure, which may account for certain pathological features of multiple sclerosis, including demyelination, oligodendrocyte apoptosis and astrocyte dysfunction [9]. In RRMS, activated RNS, which can cause nitrosative stress, may lead to myelin destruction. Due to the elevated expression of NOS in activated microglia and infiltrated macrophages, nitric oxide (NO) production is significantly increased in the inflammatory region. Evidence suggests that NO contributes to tissue injury, blood-brain barrier (BBB) breakdown, axonal degeneration and plaque formation. On the other hand, NO may help to control the immune response via several immunoregulatory processes. It is a marker of nitrosative stress in cells and tissues and also a relatively specific marker of oxidative damage, mediated by peroxynitrite, which is formed by the reaction of superoxide with NO [10, 11]. The central nervous system (CNS) is particularly susceptible to oxidative stress due to the high rate of oxygen utilization, a relatively poor antioxidant defence system [12]. In recent years, the role of methylprednisolone (MP) in the RRMS treatment has gained strength. Due to their strong anti-inflammatory action, they are used most effectively to overcome and shorten the duration of neurological symptoms during relapses. MP activity consists mainly of the induction of specific inflammation suppressing genes and repression of genes coding proteins involved in immune and inflammatory reactions. MP therapy results in inhibition of inflammatory reactions, sealing of BBB and decreasing the magnetic resonance imaging (MRI) number of gadolinium up-taking foci [13].
Blood Detection The following items were detected with biochemical methods. Cholesterol (mg/dl), Triglycerides (mg/dl), HDL (mg/dl), LDL (mg/dl), CRP (mg/l), ESR (mm/h). Erythrocytes, WBC (103/μl), Neurophil (103/μl), Basophil (%), Eosinophil (%), Lymphocytes (%), Uric acid (mg/dl), Albumin (mg/dl). The MPO (myeloperoxidase) was determined with MPO Elisa kits. Statistical Analysis The data were expressed as mean ± SD, statistical analysis was performed with one-way analysis of variance (ANOVA), followed by LSD’s post hoc test. A p value of less than 0.05 (* p < 0.05) was considered statistically significant. The statistical analysis was performed using the SPSS v. 17 statistical software packages (SPSS Inc., Chicago, Ill., USA).
Results
Demographic data for study groups are shown in table 1. In addition, basic metabolic profile/blood chemistries did not show significant differences (table 1). RR-MS patients displayed a nonsignificant reduction in uric acid levels (table 1). But in CRP (It is a biomarker of systemic inflammation), there is a minor difference between control and RRMS patients. The value of control is 35.4 ± 3.4 mg/l, while in RRMS patients is 8.4 ± 3.03 mg/l. The value of CRP in MS patients is much lower than that of the control patients. MPO (myeloperoxidase) plays an important role in neutrophil microbicidal action through catalyzing chlorideion oxidation to hypochlorous acid, which is a potent antimicrobial agent. MPO possesses potent pro inflammatory properties and may contribute directly to tissue injury. In addition, MPO is shown to be involved in pathogenesis of lung cancer, Alzheimer’s disease and multiple sclerosis [17]. In our cases, the level of MPO increased significantly in RRMS patients by 120% (** p < 0.01).
Table 1. Subjects’ characteristics and blood chemistries in relaps-
ing-remitting multiple sclerosis (RR-MS) Control group (n = 20) Gender (male/female) Age, years Disease duration, years Scale of EDSS (subjects, n)
