J Mol Neurosci (2014) 54:767–773 DOI 10.1007/s12031-014-0366-8
Concentration of Soluble Adhesion Molecules in Cerebrospinal Fluid and Serum of Epilepsy Patients Jing Luo & Wei Wang & Zhiqin Xi & Chen dan & Liang Wang & Zheng Xiao & Xuefeng Wang
Received: 22 December 2013 / Accepted: 26 June 2014 / Published online: 8 July 2014 # Springer Science+Business Media New York 2014
Abstract Mounting evidence supports the involvement of brain inflammation and the associated blood–brain barrier damage from which spontaneous and recurrent seizures originate. Detection of the soluble form of adhesion molecules (AM) has also been proven to predict outcomes in central nervous system (CNS) disorders. A recent study has shown that expression of AM in brain vessels was upregulated 24 h after kainic acid (KA) induced seizures. The aim of the present study was to investigate soluble AM levels in the cerebrospinal fluid (CSF) and serum of epilepsy patients. Paired CSF and serum samples were analyzed by sandwich enzymelinked immunosorbent assay (ELISA) to determine the concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1). Increased serum concentrations of sICAM-1 were present in epileptic patients (41 localization-related of unknown etiology, 19 idiopathic generalized). Serum sICAM-1 level in drug-refractory epilepsy was elevated as compared to new diagnosis epilepsy and drug-responsive epilepsy. CSF sVCAM-1 and serum sVCAM-1 concentrations in the epilepsy group were higher as compared to the neurosis group. Moreover, CSF sVCAM-1 and serum sVCAM-1 concentrations in drug-refractory epilepsy were raised as compared to drug-responsive epilepsy and new diagnosis epilepsy. However, there were no significant differences in concentrations of Jing Luo and Wei Wang contributed equally to this work. J. Luo : W. Wang : Z. Xi : L. Wang : Z. Xiao : X. Wang (*) Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China e-mail: [email protected] C. dan Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China
CSF sICAM-1 between the epilepsy and neurosis groups. Our results suggest that sVCAM-1 and sICAM-1 could play an important role in the drug-refractory epilepsy. Keywords sICAM-1 . sVCAM-1 . Epilepsy . Blood–brain barrier
Introduction Epilepsy is a chronic neurological disorder that affects approximately 1 % of the world population, but the mechanisms involved in the pathogenesis of epilepsy, are still not clearly understood (Bialer and White 2010, Kwan et al. 2011). In the last decade, cytokines and vascular alterations have been discussed in relation to the pathogenesis of epilepsy, suggesting brain inflammation as a crucial etiopathogenetic mechanism of epilepsy that could be targeted to control seizures (Librizzi et al. 2012). The emerging roles of leukocytes and leukocyte adhesion mechanisms in seizure generation provides insights into the mechanisms of brain damage, and could contribute to the development of novel therapeutic strategies in epilepsy (Fabene et al. 2008). A recent study has shown that in seizures induced by kainic acid, expression of adhesion molecule (AM) in brain vessels was upregulated 24 h after the onset of seizure (Fabene et al. 2013). AM can be released from cerebral endothelial cell membranes upon cytokine stimulation and detected in body fluids. Increased levels of these soluble AM have been detected in several neurological diseases such as multiple sclerosis and myasthenia gravis (Khoury et al. 1999, Tesa et al. 2000, Correale and Bassani Molinas 2003). AM of these family members, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are known to be expressed in brain microvessel endothelia cells (EC), astrocytes, and microglia (Lee and Benveniste 1999). Soluble
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J Mol Neurosci (2014) 54:767–773
intercellular adhesion molecule-1 (sICAM-1) has been found in such body fluids such as serum, cerebrospinal fluid (CSF), synovial fluid, and sputum and represents a circulating form of ICAM-1 (Witkowska and Borawska 2004). VCAM-1 is another member of the immunoglobulin super-gene family and soluble vascular cell adhesion molecule-1 (sVCAM-1) also has been found in serum and CSF (Kraus et al. 2002). Brain inflammation is known to be involved in blood–brain barrier damage, which leads to spontaneous and recurrent seizures (Vezzani et al. 2013a, b). Detection of the soluble form of AM has also been shown to predict outcomes in central nervous system (CNS) disorders (Ma et al. 2013). In this study, we will detect the sICAM-1 and sVCAM-1 levels of expression in the serum and CSF of epilepsy patients and association with clinical pathogenesis by sandwich enzymelinked immunosorbent assays (ELISA).
Materials and Methods Subjects A total of 60 patients (34 males and 26 females), between 13 to 65 years of age (mean±S.D., 35.2±14.8 years), were recruited from the Epilepsy Clinic of the Department of Neurology of the First Affiliated Hospital of Chongqing Medical University. All patients went through a comprehensive clinical examination, including a detailed medical history, neurological and neuropsychological examinations, and an electroencephalogram (EEG) study. Cranial magnetic resonance imaging (MRI) or computed tomography (CT) scans found no progressive lesions in the CNS. Epilepsy was diagnosed as idiopathic epilepsy and classified according to the criteria proposed by the International League against Epilepsy (ILAE) in 2001(Seino 2006). In our study, 41 out of 60 epilepsy patients had complex partial seizures or secondarily generalized tonic–clonic seizures (2-GTCS). These patients were diagnosed as localization-related epilepsy (LRE) of unknown etiology. The other 19 out of 60 epilepsy patients had absence or primarily GTCS, and these patients were diagnosed as Idiopathic generalized epilepsy (IGE). EEG was used
to help determine the diagnosis of epilepsy, but not as a basis for the classification of primary or secondary epilepsy. In the epilepsy group, 18 of patients (8 males and 10 females) had a seizure history of more than 3 years, and were on regular treatment with at least three types of common antiepileptic drugs (AEDs), but their seizures were not controlled. This group was defined as the drug-refractory epilepsy (DRE) subgroup. The second epilepsy subgroup consisted of 18 patients (11 males and 7 females) who had at least one seizure attack before diagnosis and did not take any AEDs. This group was defined as the new-diagnosis epilepsy (NDE) subgroup according to the diagnostic methods of the ILAE. The third epilepsy subgroup comprised of 24 patients (15 males and 9 females) who had been seizure-free for more than 2 years after taking one type of AEDs. This group was defined as the drug responsive-epilepsy (DPE) subgroup. A total of 17 controls (9 males and 8 females), between 14 to 70 years of age (mean± S.D., 35.8± 18.3 years) were included. The control group consisted of patients with neurosis, which includes somatization and anxiety disorders. Patients of the control group had no evidence of neuropsychological or neurological disorders or any past history of seizures. No progressive lesions in the CNS were found by neuroradiological study including brain MRI or CT scans. Tables 1, 2 and 3 show the clinical features of epilepsy patients and controls.
Sample Collection and Storage CSF and venous blood samples were collected following the procedure in our lab using a previously described procedure (Xiao et al. 2009). All epilepsy patients in this study had no seizure within 24 h before sample collection. Two milliliters of CSF and 5 ml of venous blood samples were collected from each subject in our study. The blood samples were centrifuged at 3,000g for 15 min to collect the serum, which was then aliquoted and frozen at −80 °C until the aliquots were processed as one batch at in the end of the study. CSF samples were centrifuged at 2,000g for 10 min at 4 °C and frozen at −80 °C until the samples were processed as one batch at the end of the study.
Table 1 Clinical feature of the subjects Clinical variable
DRE (n=18)
NDE (n=18)
DPE (n=24)
Neurosis (n=17)
p value*
Age (years) Mean±SD
36.7±15.3
29.9±13.8
38.1±14.6
35.8±18.3
0.376
Range Male/female ratio
13~65 8/10
15~60 11/7
16~65 15/9
14~70 9/8
0.650
DRE drug-refractory epilepsy subgroup, NDE new diagnosis epilepsy subgroup, DPE drug-response epilepsy subgroup *p
Concentration of Soluble Adhesion Molecules in Cerebrospinal Fluid and Serum of Epilepsy Patients Jing Luo & Wei Wang & Zhiqin Xi & Chen dan & Liang Wang & Zheng Xiao & Xuefeng Wang
Received: 22 December 2013 / Accepted: 26 June 2014 / Published online: 8 July 2014 # Springer Science+Business Media New York 2014
Abstract Mounting evidence supports the involvement of brain inflammation and the associated blood–brain barrier damage from which spontaneous and recurrent seizures originate. Detection of the soluble form of adhesion molecules (AM) has also been proven to predict outcomes in central nervous system (CNS) disorders. A recent study has shown that expression of AM in brain vessels was upregulated 24 h after kainic acid (KA) induced seizures. The aim of the present study was to investigate soluble AM levels in the cerebrospinal fluid (CSF) and serum of epilepsy patients. Paired CSF and serum samples were analyzed by sandwich enzymelinked immunosorbent assay (ELISA) to determine the concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1). Increased serum concentrations of sICAM-1 were present in epileptic patients (41 localization-related of unknown etiology, 19 idiopathic generalized). Serum sICAM-1 level in drug-refractory epilepsy was elevated as compared to new diagnosis epilepsy and drug-responsive epilepsy. CSF sVCAM-1 and serum sVCAM-1 concentrations in the epilepsy group were higher as compared to the neurosis group. Moreover, CSF sVCAM-1 and serum sVCAM-1 concentrations in drug-refractory epilepsy were raised as compared to drug-responsive epilepsy and new diagnosis epilepsy. However, there were no significant differences in concentrations of Jing Luo and Wei Wang contributed equally to this work. J. Luo : W. Wang : Z. Xi : L. Wang : Z. Xiao : X. Wang (*) Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China e-mail: [email protected] C. dan Department of Neurosurgery, The First Affiliated Hospital of Chongqing Medical University, 1 You Yi Road, Chongqing 400016, China
CSF sICAM-1 between the epilepsy and neurosis groups. Our results suggest that sVCAM-1 and sICAM-1 could play an important role in the drug-refractory epilepsy. Keywords sICAM-1 . sVCAM-1 . Epilepsy . Blood–brain barrier
Introduction Epilepsy is a chronic neurological disorder that affects approximately 1 % of the world population, but the mechanisms involved in the pathogenesis of epilepsy, are still not clearly understood (Bialer and White 2010, Kwan et al. 2011). In the last decade, cytokines and vascular alterations have been discussed in relation to the pathogenesis of epilepsy, suggesting brain inflammation as a crucial etiopathogenetic mechanism of epilepsy that could be targeted to control seizures (Librizzi et al. 2012). The emerging roles of leukocytes and leukocyte adhesion mechanisms in seizure generation provides insights into the mechanisms of brain damage, and could contribute to the development of novel therapeutic strategies in epilepsy (Fabene et al. 2008). A recent study has shown that in seizures induced by kainic acid, expression of adhesion molecule (AM) in brain vessels was upregulated 24 h after the onset of seizure (Fabene et al. 2013). AM can be released from cerebral endothelial cell membranes upon cytokine stimulation and detected in body fluids. Increased levels of these soluble AM have been detected in several neurological diseases such as multiple sclerosis and myasthenia gravis (Khoury et al. 1999, Tesa et al. 2000, Correale and Bassani Molinas 2003). AM of these family members, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) are known to be expressed in brain microvessel endothelia cells (EC), astrocytes, and microglia (Lee and Benveniste 1999). Soluble
768
J Mol Neurosci (2014) 54:767–773
intercellular adhesion molecule-1 (sICAM-1) has been found in such body fluids such as serum, cerebrospinal fluid (CSF), synovial fluid, and sputum and represents a circulating form of ICAM-1 (Witkowska and Borawska 2004). VCAM-1 is another member of the immunoglobulin super-gene family and soluble vascular cell adhesion molecule-1 (sVCAM-1) also has been found in serum and CSF (Kraus et al. 2002). Brain inflammation is known to be involved in blood–brain barrier damage, which leads to spontaneous and recurrent seizures (Vezzani et al. 2013a, b). Detection of the soluble form of AM has also been shown to predict outcomes in central nervous system (CNS) disorders (Ma et al. 2013). In this study, we will detect the sICAM-1 and sVCAM-1 levels of expression in the serum and CSF of epilepsy patients and association with clinical pathogenesis by sandwich enzymelinked immunosorbent assays (ELISA).
Materials and Methods Subjects A total of 60 patients (34 males and 26 females), between 13 to 65 years of age (mean±S.D., 35.2±14.8 years), were recruited from the Epilepsy Clinic of the Department of Neurology of the First Affiliated Hospital of Chongqing Medical University. All patients went through a comprehensive clinical examination, including a detailed medical history, neurological and neuropsychological examinations, and an electroencephalogram (EEG) study. Cranial magnetic resonance imaging (MRI) or computed tomography (CT) scans found no progressive lesions in the CNS. Epilepsy was diagnosed as idiopathic epilepsy and classified according to the criteria proposed by the International League against Epilepsy (ILAE) in 2001(Seino 2006). In our study, 41 out of 60 epilepsy patients had complex partial seizures or secondarily generalized tonic–clonic seizures (2-GTCS). These patients were diagnosed as localization-related epilepsy (LRE) of unknown etiology. The other 19 out of 60 epilepsy patients had absence or primarily GTCS, and these patients were diagnosed as Idiopathic generalized epilepsy (IGE). EEG was used
to help determine the diagnosis of epilepsy, but not as a basis for the classification of primary or secondary epilepsy. In the epilepsy group, 18 of patients (8 males and 10 females) had a seizure history of more than 3 years, and were on regular treatment with at least three types of common antiepileptic drugs (AEDs), but their seizures were not controlled. This group was defined as the drug-refractory epilepsy (DRE) subgroup. The second epilepsy subgroup consisted of 18 patients (11 males and 7 females) who had at least one seizure attack before diagnosis and did not take any AEDs. This group was defined as the new-diagnosis epilepsy (NDE) subgroup according to the diagnostic methods of the ILAE. The third epilepsy subgroup comprised of 24 patients (15 males and 9 females) who had been seizure-free for more than 2 years after taking one type of AEDs. This group was defined as the drug responsive-epilepsy (DPE) subgroup. A total of 17 controls (9 males and 8 females), between 14 to 70 years of age (mean± S.D., 35.8± 18.3 years) were included. The control group consisted of patients with neurosis, which includes somatization and anxiety disorders. Patients of the control group had no evidence of neuropsychological or neurological disorders or any past history of seizures. No progressive lesions in the CNS were found by neuroradiological study including brain MRI or CT scans. Tables 1, 2 and 3 show the clinical features of epilepsy patients and controls.
Sample Collection and Storage CSF and venous blood samples were collected following the procedure in our lab using a previously described procedure (Xiao et al. 2009). All epilepsy patients in this study had no seizure within 24 h before sample collection. Two milliliters of CSF and 5 ml of venous blood samples were collected from each subject in our study. The blood samples were centrifuged at 3,000g for 15 min to collect the serum, which was then aliquoted and frozen at −80 °C until the aliquots were processed as one batch at in the end of the study. CSF samples were centrifuged at 2,000g for 10 min at 4 °C and frozen at −80 °C until the samples were processed as one batch at the end of the study.
Table 1 Clinical feature of the subjects Clinical variable
DRE (n=18)
NDE (n=18)
DPE (n=24)
Neurosis (n=17)
p value*
Age (years) Mean±SD
36.7±15.3
29.9±13.8
38.1±14.6
35.8±18.3
0.376
Range Male/female ratio
13~65 8/10
15~60 11/7
16~65 15/9
14~70 9/8
0.650
DRE drug-refractory epilepsy subgroup, NDE new diagnosis epilepsy subgroup, DPE drug-response epilepsy subgroup *p
