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Altered Expression of CX3CL1 in Patients with Epilepsy and in a Rat Model

  • Yali Xu
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Kebin Zeng
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Yanbing Han
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Liang Wang
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Dan Chen
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Zhiqin Xi
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Heng Wang
    Affiliations
    Department of Neurology, The Second Affiliated Hospital, Chongqing Medical University, Chongqing, China
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  • Xuefeng Wang
    Correspondence
    Address reprint requests to Xuefeng Wang, M.D. or Guojun Chen, M.D., Ph.D., Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, 1Youyi Rd, Chongqing, 400016, China
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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  • Guojun Chen
    Correspondence
    Address reprint requests to Xuefeng Wang, M.D. or Guojun Chen, M.D., Ph.D., Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, 1Youyi Rd, Chongqing, 400016, China
    Affiliations
    Department of Neurology, The First Affiliated Hospital, Chongqing Medical University, Chongqing Key Laboratory of Neurology, Chongqing, China
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      Chemokine C-X3-C motif ligand 1 (CX3CL1, alias fractalkine), is highly expressed in the central nervous system and participates in inflammatory responses. Recent studies indicated that inflammatory processes within the brain constitute a common and crucial mechanism in the pathophysiological characteristics of epilepsy. This study investigated the expression pattern of CX3CL1 in epilepsy and its relationship with neuronal loss. Double immunolabeling, IHC, and immunoblotting results showed that CX3CL1 expression was up-regulated in the temporal neocortex of patients with temporal lobe epilepsy. In a rat model of epilepsy, CX3CL1 up-regulation began 6 hours after epilepsy, with relatively high expression for 60 days. In addition, ELISA revealed that the concentrations of CX3CL1 in cerebrospinal fluid and serum were higher in epileptic patients than in patients with neurosis but lower than in patients with inflammatory neurological diseases. Moreover, H&E staining demonstrated significant neuronal loss in the brains of epileptic patients and in the rat model. Finally, the expression of tumor necrosis factor–related apoptosis-inducing ligand was significantly increased in both patients and the animal model, suggesting that tumor necrosis factor–related apoptosis-inducing ligand may play a role in CX3CL1-induced cell death. Thus, our results indicate that CX3CL1 may serve as a possible biomarker of brain inflammation in epileptic patients.
      Epilepsy is characterized by an enduring predisposition to seizures and by emotional and cognitive dysfunctions.
      • Duncan J.S.
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      Adult epilepsy.
      This disorder affects approximately 50 million people worldwide and, therefore, is one of the most common neurological disorders.
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      The role of inflammation in epilepsy.
      During the past 10 years, an increasing body of clinical and experimental evidence has provided strong support to the hypothesis that inflammatory processes within the brain might contribute to the pathophysiological characteristics of epilepsy.
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      The role of inflammation in epilepsy.
      For instance, an increased expression of inflammatory markers (high-mobility group box-1,
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      Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures.
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      Expression of monocyte chemoattractant protein-1 in brain tissue of patients with intractable epilepsy.
      NF-κB,
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      Inflammatory reactions in human medial temporal lobe epilepsy with hippocampal sclerosis.
      and transforming growth factor-β type 1 receptor
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      Increased expression of TGFbeta type I receptor in brain tissues of patients with temporal lobe epilepsy.
      ) has been observed in surgically resected temporal tissues from patients with temporal lobe epilepsy (TLE).
      • Vezzani A.
      • Granata T.
      Brain inflammation in epilepsy: experimental and clinical evidence.
      A similar inflammatory reaction has also been reported in experimental models of TLE. In particular, an increase of pro-inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-α) has been detected in the rat hippocampus, starting within the first hour after the induction of a status epilepticus (SE) and lasting for several days.
      • Ravizza T.
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      • Vezzani A.
      Innate and adaptive immunity during epileptogenesis and spontaneous seizures: evidence from experimental models and human temporal lobe epilepsy.
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      Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus.
      Chemokines are cytokines that orchestrate the traffic of leukocytes throughout the body.
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      • Emilie D.
      Deregulation of the expression of the fractalkine/fractalkine receptor complex in HIV-1-infected patients.
      CX3CL1 (chemokine C-X3-C motif ligand)/fractalkine, which is expressed in the central nervous system, participates in inflammatory responses in many brain disorders.
      • Kastenbauer S.
      • Koedel U.
      • Wick M.
      • Kieseier B.C.
      • Hartung H.P.
      • Pfister H.W.
      CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system.
      • Pabon M.M.
      • Bachstetter A.D.
      • Hudson C.E.
      • Gemma C.
      • Bickford P.C.
      CX3CL1 reduces neurotoxicity and microglial activation in a rat model of Parkinson's disease.
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      • Jun M.
      • Guo-Hua W.
      • Ya-Ling H.
      • Hua-Wei L.
      • Ding-Fang C.
      New evidences for fractalkine/CX3CL1 involved in substantia nigral microglial activation and behavioral changes in a rat model of Parkinson's disease.
      CX3CL1 is the fourth chemokine type (CX3C motif), with three amino acid residues between the first and second cysteine.
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      • Rossi D.
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      • Schall T.J.
      A new class of membrane-bound chemokine with a CX3C motif.
      Unlike other chemokines, CX3CL1 is produced as a membrane-bound form presented at the cell surface by a mucinlike stalk. CX3CL1 can be released as a soluble form after proteolytic cleavage and is primarily localized in neurons and endothelial cells.
      • Beck G.C.
      • Yard B.A.
      • Breedijk A.J.
      • Van Ackern K.
      • Van Der Woude F.J.
      Release of CXC-chemokines by human lung microvascular endothelial cells (LMVEC) compared with macrovascular umbilical vein endothelial cells.
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      • McNamara R.K.
      • Streit W.J.
      • Salafranca M.N.
      • Adhikari S.
      • Thompson D.A.
      • Botti P.
      • Bacon K.B.
      • Feng L.
      Role for neuronally derived fractalkine in mediating interactions between neurons and CX3CR1-expressing microglia.
      • Meucci O.
      • Fatatis A.
      • Simen A.A.
      • Miller R.J.
      Expression of CX3CR1 chemokine receptors on neurons and their role in neuronal survival.
      • Nishiyori A.
      • Minami M.
      • Ohtani Y.
      • Takami S.
      • Yamamoto J.
      • Kawaguchi N.
      • Kume T.
      • Akaike A.
      • Satoh M.
      Localization of fractalkine and CX3CR1 mRNAs in rat brain: does fractalkine play a role in signaling from neuron to microglia?.
      • Rancan M.
      • Bye N.
      • Otto V.I.
      • Trentz O.
      • Kossmann T.
      • Frentzel S.
      • Morganti-Kossmann M.C.
      The chemokine fractalkine in patients with severe traumatic brain injury and a mouse model of closed head injury.
      Because of its characteristic structure, multiple properties, and cellular expression pattern, it has been hypothesized that CX3CL1 is involved in the intercellular communication among neurons, microglia, and endothelial cells, as well as in extravasation of leukocytes after brain injury and inflammatory central nervous system diseases.
      • Kastenbauer S.
      • Koedel U.
      • Wick M.
      • Kieseier B.C.
      • Hartung H.P.
      • Pfister H.W.
      CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system.
      • Rancan M.
      • Bye N.
      • Otto V.I.
      • Trentz O.
      • Kossmann T.
      • Frentzel S.
      • Morganti-Kossmann M.C.
      The chemokine fractalkine in patients with severe traumatic brain injury and a mouse model of closed head injury.
      • Hughes P.M.
      • Botham M.S.
      • Frentzel S.
      • Mir A.
      • Perry V.H.
      Expression of fractalkine (CX3CL1) and its receptor, CX3CR1, during acute and chronic inflammation in the rodent CNS.
      Although CX3CL1 is likely to contribute to the inflammatory processes in epilepsy, to our knowledge, no study has addressed the expression of CX3CL1 in brain tissue of epileptic patients and rat models.
      In this study, we investigated the expression of CX3CL1 in the temporal neocortex of patients with TLE, as well as the concentrations of CX3CL1 in cerebrospinal fluid (CSF) and serum from epileptic patients. To extend the results gained through the analysis of human tissues, the expression of CX3CL1 was investigated in the hippocampus and adjacent cortex of a rat model of TLE at different time points after epilepsy. The neuronal loss and expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) after epilepsy were also examined.

      Materials and Methods

      Human Brain Tissue and Clinical Data

      Thirty patients undergoing surgery for medically intractable TLE and 15 cases of nonepilepsy control were included in this study. All brain tissue was chosen at random from our epilepsy brain tissue bank. The brain tissue bank was reported in our previous studies.
      • Li J.M.
      • Wang X.F.
      • Xi Z.Q.
      • Gong Y.
      • Liu F.Y.
      • Sun J.J.
      • Wu Y.
      • Luan G.M.
      • Wang Y.P.
      • Li Y.L.
      • Zhang J.G.
      • Lu Y.
      • Li H.W.
      Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy.
      • Pan Y.
      • Liu G.
      • Fang M.
      • Shen L.
      • Wang L.
      • Han Y.
      • Shen D.
      • Wang X.
      Abnormal expression of netrin-G2 in temporal lobe epilepsy neurons in humans and a rat model.
      • Fang M.
      • Liu G.W.
      • Pan Y.M.
      • Shen L.
      • Li C.S.
      • Xi Z.Q.
      • Xiao F.
      • Wang L.
      • Chen D.
      • Wang X.F.
      Abnormal expression and spatiotemporal change of Slit2 in neurons and astrocytes in temporal lobe epileptic foci: a study of epileptic patients and experimental animals.
      All procedures were performed with the formal consent of the patients or legal next of kin and were approved by the ethics committees of the respective institutions, according to the Declaration of Helsinki. Our study protocol complied with the guidelines for the conduct of research involving human subjects, as established by the NIH and the Committee on Human Research at Chongqing Medical University, Chongqing, China.
      Presurgical assessment comprised a detailed history and neurological examination, interictal and ictal electroencephalographic studies, neuropsychological testing, and neuroradiological studies. At surgery, all patients with TLE were refractory to maximal doses of three or more anti-epileptic drugs, including carbamazepine, clonazepam, lamotrigine, phenobarbital, phenytoin, topiramate, and valproic acid. All tissue blocks were from resections performed for strictly therapeutic purposes. After lesion resection, electrodes for intraoperative electrocorticography were placed on the remaining edge of the tissue to ensure that the lesion had been completely resected. Table 1 summarizes the clinical features. Pathological diagnoses included neuron loss (n = 24) and neuron loss and neocortical gliosis (n = 6).
      Table 1Comparison of Clinical Data in the Patients with TLE and the Nonepileptic Controls
      Clinical variableTLE group (n = 30)Control group (n = 15)P value
      P values were computed using an independent-sample t-test (age) or a χ2 test (male/female ratio). P < 0.05 was considered significant.
      Age (years)
       Mean ± SD25.1 ± 12.323.2 ± 11.50.603
       Range8–5815–50
      Epilepsy onset (years)
       Mean ± SD13.8 ± 9.8NANA
       Range1–38NA
      Epilepsy duration
      Epilepsy duration was calculated as the time between epilepsy onset (onset of habitual seizures) and surgery.
      (years)
       Mean ± SD11.0 ± 7.7NANA
       Range1–29NA
      Seizure frequency (no./month)
       Mean ± SD10.1 ± 6.9NANA
       Range3–30NA
      Seizure type
       CPS8NANA
       SGS17NA
       CPS and SGS5NA
      Male/female ratio16:149:60.757
      CPS, complex partial seizures; NA, not applicable; SGS, secondary generalized seizure.
      low asterisk P values were computed using an independent-sample t-test (age) or a χ2 test (male/female ratio). P < 0.05 was considered significant.
      Epilepsy duration was calculated as the time between epilepsy onset (onset of habitual seizures) and surgery.
      For comparison, we obtained 15 histological normal temporal neocortex samples from individuals treated for increased intracranial pressure due to head trauma from a motor vehicle crash, who later died from these injuries. Brain samples from controls were taken only when the brain tissues were immediately exposed outside the skull after severe head injury, which were no longer available for rescue, or immediately after death. A consent form was signed by either patient when possible or close relative(s) before study. A conventional neuropathological examination revealed no signs of central nervous system disease. The mean ± SD age of the control group was 23.17 ± 11.52 years (range, 15 to 50 years). These subjects had no history of epilepsy or exposure to anti-epileptic drugs. There were no significant differences in age, sex, or topography of the studied tissues between TLE and control tissues.

      CSF and Serum

      Patient Material

      All CSF and blood samples were obtained from the Department of Neurology (The First Affiliated Hospital, Chongqing Medical University).
      • Wang L.
      • Pan Y.
      • Chen D.
      • Xiao Z.
      • Xi Z.
      • Xiao F.
      • Wang X.
      Tetranectin is a potential biomarker in cerebrospinal fluid and serum of patients with epilepsy.
      • Xiao F.
      • Chen D.
      • Lu Y.
      • Xiao Z.
      • Guan L.F.
      • Yuan J.
      • Wang L.
      • Xi Z.Q.
      • Wang X.F.
      Proteomic analysis of cerebrospinal fluid from patients with idiopathic temporal lobe epilepsy.
      The CSF samples from 39 living epileptic patients were collected by lumbar puncture during conscious sedation. Patients were completely seizure free for >24 hours before CSF collection. The control group comprised 27 control subjects with inflammatory neurological diseases (bacterial meningitis, Bell's palsy, Guillain-Barre syndrome, and viral meningitis) and 8 control subjects with neurosis. Bacterial meningitis, Bell's palsy, Guillain-Barre syndrome, and viral meningitis were all considered inflammatory neurological diseases.
      • Kastenbauer S.
      • Koedel U.
      • Wick M.
      • Kieseier B.C.
      • Hartung H.P.
      • Pfister H.W.
      CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system.
      • Sainaghi P.P.
      • Collimedaglia L.
      • Alciato F.
      • Leone M.A.
      • Naldi P.
      • Molinari R.
      • Monaco F.
      • Avanzi G.C.
      The expression pattern of inflammatory mediators in cerebrospinal fluid differentiates Guillain-Barre syndrome from chronic inflammatory demyelinating polyneuropathy.
      • Nyati K.K.
      • Prasad K.N.
      • Rizwan A.
      • Verma A.
      • Paliwal V.K.
      TH1 and TH2 response to Campylobacter jejuni antigen in Guillain-Barre syndrome.
      • Salinas R.A.
      • Alvarez G.
      • Daly F.
      • Ferreira J.
      Corticosteroids for Bell's palsy (idiopathic facial paralysis).
      Neurosis is also termed neurotic disorders, which include somatization and anxiety disorders. Patients who presented with pain (eg, headaches, pain in the extremities, and back pain) or pseudoneurological symptoms (eg, difficulty swallowing, pseudoseizures, double or blurred vision, and blindness) were recruited in the inpatient department, where computed tomography, magnetic resonance imaging, or CSF were further performed, as indicated, to exclude possible underlying organic abnormalities. These patients were finally diagnosed as having neurosis or neurotic disorders and were used as controls in this study. Relevant clinical characteristics are summarized in Table 2, Table 3.
      Table 2Clinical Features of the 74 Patients in the CSF Study
      Clinical variableEpilepsy group (n = 39)Inflammation group (n = 27)Neurosis group (n = 8)P value
      P values were computed using a one-way analysis of variance (age) or a χ2 exact test (male/female ratio). P < 0.05 was considered significant.
      Age (years)
       Mean ± SD29.4 ± 11.334.8 ± 12.633.4 ± 12.30.190
       Range15–6016–6018–55
      Male/female ratio22:1713:144:40.793
      low asterisk P values were computed using a one-way analysis of variance (age) or a χ2 exact test (male/female ratio). P < 0.05 was considered significant.
      Table 3Clinical Features of the Epileptic Patients in the CSF and Serum Studies
      Clinical variableCSF group (n = 39)Serum group (n = 43)
      Age (years)
       Mean ± SD29.4 ± 11.330.0 ± 11.7
       Range15–6016–60
      Seizure frequency (no./month)
       Mean ± SD6.6 ± 5.26.9 ± 4.8
       Range0.05–200.05–20
      Seizure frequency (no./month)
       <52119
       5–101116
       10–2078
      Seizure duration (years)
      Epilepsy duration was calculated as the time between epilepsy onset (onset of habitual seizures) and sample collection.
       <11215
       1–51111
       >51617
      Seizure type
       CPS1517
       GTCS79
       SGS1717
      Male/female ratio22:1723:20
      Data are given as number of patients unless otherwise indicated.
      CPS, complex partial seizure; GTCS, generalized tonic-clonic seizure; SGS, secondary generalized seizure.
      low asterisk Epilepsy duration was calculated as the time between epilepsy onset (onset of habitual seizures) and sample collection.
      Serum samples were obtained from 43 living epileptic patients (patients were also completely seizure free for >24 hours before blood collection), 26 control subjects with inflammatory neurological diseases (bacterial meningitis, Bell's palsy, Guillain-Barre syndrome, and viral meningitis), and 9 control subjects with neurosis. Relevant clinical characteristics are summarized in Table 3, Table 4.
      Table 4Clinical Features of the 78 Patients in the Serum Study
      Clinical variableEpilepsy group (n = 43)Inflammation group (n = 26)Neurosis group (n = 9)P value
      P values were computed using a one-way analysis of variance (age) or a χ2 exact test (male/female ratio). P < 0.05 was considered significant.
      Age (years)
       Mean ± SD30.0 ± 11.731.3 ± 11.531.7 ± 12.60.873
       Range16–6016–6018–55
      Male/female ratio23:2012:144:50.789
      low asterisk P values were computed using a one-way analysis of variance (age) or a χ2 exact test (male/female ratio). P < 0.05 was considered significant.
      In the CSF and serum study, 40 patients (21 with epilepsy, 11 with inflammatory neurological diseases, and 8 with neurosis) provided both CSF and serum samples. In other words, peripheral venous blood and lumbar CSF samples were both collected from the same patients. The clinical characteristics are summarized in Table 5.
      Table 5Clinical Features of the 40 Patients in the CSF/Serum Study
      Clinical variableEpilepsy group (n = 21)Inflammation group (n = 11)Neurosis group (n = 8)P value
      P values were computed using a one-way analysis of variance (age) or a χ2 exact test (male/female ratio). P < 0.05 was considered significant.
      Age (years)
       Mean ± SD26.9 ± 10.033. 5 ± 14.533.4 ± 12.30.232
       Range16–5416–6018–55
      Male/female ratio12:94:74:40.536
      low asterisk P values were computed using a one-way analysis of variance (age) or a χ2 exact test (male/female ratio). P < 0.05 was considered significant.
      The study was approved by the Committee on Human Research at Chongqing Medical University, and written informed consent was obtained from all participants.

      CSF and Serum Studies

      The CSF and venous blood samples drawn in EDTA tubes were cooled immediately after sampling, centrifuged, and stored frozen at −80° until analysis, as previously described.
      • Wang L.
      • Pan Y.
      • Chen D.
      • Xiao Z.
      • Xi Z.
      • Xiao F.
      • Wang X.
      Tetranectin is a potential biomarker in cerebrospinal fluid and serum of patients with epilepsy.
      CX3CL1 was measured with commercially available ELISA kits (Human CX3CL1/Fractalkine Immunoassay, number DCX310; R&D Systems, Minneapolis, MN). Values were calculated from a standard curve generated for each ELISA result. Samples were not diluted, and results were standardized according to previously established protein concentrations, with the final concentration expressed as ng/mL protein.

      Rat Model of Epilepsy

      All animal procedures were approved by the Commission of Chongqing Medical University for ethics of experiments on animals and were conducted in accordance with international standards.
      The rat model was made as previously reported in our laboratory.
      • Pan Y.
      • Liu G.
      • Fang M.
      • Shen L.
      • Wang L.
      • Han Y.
      • Shen D.
      • Wang X.
      Abnormal expression of netrin-G2 in temporal lobe epilepsy neurons in humans and a rat model.
      • Fang M.
      • Liu G.W.
      • Pan Y.M.
      • Shen L.
      • Li C.S.
      • Xi Z.Q.
      • Xiao F.
      • Wang L.
      • Chen D.
      • Wang X.F.
      Abnormal expression and spatiotemporal change of Slit2 in neurons and astrocytes in temporal lobe epileptic foci: a study of epileptic patients and experimental animals.
      Healthy adult male Sprague-Dawley rats (n = 49) from Chongqing Medical University Laboratory Animal Center, weighing 200 to 250 g, were randomly divided into the normal control group (n = 7) or the experimental group (n = 42). The experimental group was randomly divided into six subgroups: 6 hours, 72 hours, 7 days, 14 days, 30 days, and 60 days after SE. Rats were injected with lithium chloride (127 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO). Approximately 18 hours later, atropine sulfate (1 mg/kg, i.p.) was administered to limit the peripheral effects of the convulsant. Thirty minutes later, SE was induced by injecting pilocarpine hydrochloride (30 mg/kg, i.p.; Sigma) in 42 rats. Pilocarpine hydrochloride was given repeatedly (10 mg/kg, i.p.) every 30 minutes until the rats developed seizures. At 6 to 11 days after pilocarpine, these rats would develop spontaneous recurrent seizures. The evoked seizures were scored according to Racine.
      • Racine R.J.
      Modification of seizure activity by electrical stimulation, I: after-discharge threshold.
      Only those rats that attained stage 4 to 5 were taken into the study. Their seizure frequency was five to eight per week. All rats were video monitored continuously starting immediately after pilocarpine injection until the day they were sacrificed. Seven control rats received the same treatment with lithium chloride and atropine sulfate, but we used saline instead of pilocarpine. At 1 hour after SE onset, we reduced the severity of convulsions with 10 mg/kg diazepam, i.p. The experimental animals were sacrificed 6 hours, 72 hours, 7 days, 14 days, 30 days, or 60 days after SE, and the hippocampus and adjacent cortex were removed for study.

      Tissue Processing

      For both human and animal tissues, one portion of resected brain tissue was immediately fixed in 10% buffered formalin for 48 hours. Tissues were then embedded in paraffin, divided into sections for immunohistochemistry (IHC; 5 μm thick) and for double-immunofluorescence labeling analysis (10 μm thick). For neuropathological evaluation, representative paraffin sections (5 μm thick) (two sections of each specimen of human and five sections of each animal) were stained with H&E. Other portions of the resected brain tissues were immediately stored in liquid nitrogen and later used for protein extraction (see the protocol for Western blot analysis). Animals were perfused transcardially with physiological saline, followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) under chloral hydrate anesthesia (0.35 g/kg, i.p.). The brains were removed and postfixed in the same fixative for 1 hour. Thereafter, the entire hippocampus and adjacent cortex were frozen and divided into sections with a cryostat (10 μm thick) for double-immunofluorescence labeling analysis.

      Double-Immunofluorescence Labeling

      Tissue sections were deparaffinized, rehydrated in a graded series of ethanol, and then incubated in H2O2 (0.3%, 15 minutes). Frozen sections were air dried on a slide warmer at 50°C for at least half an hour. For antigen retrieval, sections were treated with 10 mmol/L sodium citrate buffer (pH 6.0) and heated with a microwave oven for 20 minutes at 92°C to 98°C. Tissue was permeabilized with 0.5% Triton X-100, and then sections were incubated in 5% goat serum for 1 hour at room temperature. Sections were then incubated with a mixture of polyclonal rabbit anti-CX3CL1 antibody (1:100, catalogue number ab25088; Abcam, Cambridge, MA) and mouse anti-microtubule-associated protein-2 (MAP2) antibody (Wuhan Boster Biological Technology, Wuhan, China) or with a mixture of polyclonal rabbit anti-CX3CL1 antibody and mouse anti-glial fibrillary acidic protein (GFAP) antibody (Wuhan Boster Biological Technology) at 4°C overnight. Sections were washed and incubated with fluorescein isothiocyanate–conjugated goat anti-rabbit IgG (1:200; Zhongshan Golden Bridge Inc., Beijing, China) and tetramethylrhodamine isothiocyanate–conjugated goat anti-mouse IgG (1:200; Zhongshan Golden Bridge Inc.) in the dark for 60 minutes at room temperature, and then mounted in 50% glycerol/PBS. For TRAIL immunofluorescence, sections were incubated with a mixture of polyclonal rabbit anti-TRAIL antibody (1:50, BA1446; Wuhan Boster Biological Technology) and mouse anti-MAP2 antibody (Wuhan Boster Biological Technology) or with a mixture of polyclonal rabbit anti-TRAIL antibody and mouse anti-GFAP antibody (Wuhan Boster Biological Technology) at 4°C overnight. The secondary antibodies were fluorescein isothiocyanate–conjugated goat anti-mouse IgG (1:50; Beijing CoWin Bioscience Co, Ltd, Beijing) and tetramethylrhodamine isothiocyanate–conjugated goat anti-rabbit IgG (1:50; CoWin Bioscience Beijing, China).
      Fluoro-Jade B (FJB) staining was used to identify degenerating neurons in tissues obtained from animals.
      • van Vliet E.A.
      • da Costa Araujo S.
      • Redeker S.
      • van Schaik R.
      • Aronica E.
      • Gorter J.A.
      Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy.
      Briefly, sections were single immunofluorescence labeled of TRAIL (polyclonal rabbit anti-TRAIL antibody and tetramethylrhodamine isothiocyanate–conjugated goat anti-rabbit IgG were used) and then dried on a slide warmer. The slides were immersed in 0.06% potassium permanganate for 15 minutes and gently agitated. After rinsing in distilled water for 2 minutes, the slides were incubated for 20 minutes in 0.0004% polyanionic fluorescein derivative solution (AG310, FJB; Millipore Corporation, Billerica, MA), freshly prepared by adding 4 mL of a 0.01% stock FJB solution to 96 mL of 0.1% acetic acid, with gentle shaking in the dark. After rinsing for 1 minute in each of three changes of distilled water, the slides were dried and coverslipped with 50% glycerol/PBS. Fluorescence was detected by laser-scanning confocal microscopy (Leica Microsystems Heidelberg GmbH, Wetzlar, Germany) on an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan) equipped with a Fluoview FVX confocal scan head (Leica Microsystems Heidelberg GmbH).

      IHC Analysis

      IHC staining was conducted using the avidin-biotin-peroxidase complex method, according to established protocols or recommendations by the manufacturers. The primary antibody was rabbit anti-CX3CL1 (catalogue number ab25088; Abcam), and dilution was 1:100. The secondary antibody (Wuhan Boster Biological Technology) kit was used. For negative controls, the primary antibodies were replaced with PBS. A slide image resulting from each section was scanned and acquired by an OLYMPUS PM20 automatic microscope (Olympus, Tokyo, Japan) and a TCFY-2050 (Yuancheng Inc., Beijing, China) pathology system. Ten visual fields in each sectional image were obtained randomly (five sections in each brain). By using the Motic Med 6.0 CMIAS pathology image analysis system (Beihang Motic Inc., Beijing, China),
      • Pan Y.
      • Liu G.
      • Fang M.
      • Shen L.
      • Wang L.
      • Han Y.
      • Shen D.
      • Wang X.
      Abnormal expression of netrin-G2 in temporal lobe epilepsy neurons in humans and a rat model.
      IHC results were assessed by automatically measuring the average integrated OD that was calculated as integrated OD over field area ratio. The mean value from 10 fields in each slide image was thus collected and used to measure the differences between epilepsy and control. For TRAIL IHC, the antibody of rabbit anti-TRAIL (1:50, BA1446; Wuhan Boster Biological Technology) was used.

      Western Blot Analysis

      Proteins, 50 μg, were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore Corporation) for Western blot analysis using a Bio-Rad apparatus (Bio-Rad Laboratories, Richmond, CA). Nonspecific epitopes were blocked with 5% skim milk/Tween-20–Tris-buffered saline. The membranes were incubated overnight at 4°C with each of the following primary antibodies: rabbit anti-CX3CL1 antibody (1:300, catalogue number ab25088; Abcam), goat anti-TRAIL antibody (1:100, sc-6079; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and rabbit anti-β-actin as control antibody (1:5000; Beijing 4A Biotech Co, Ltd, Beijing). After three washes in Tween-20–Tris-buffered saline (TBST), the membranes were then treated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature and revealed with an Enhanced Chemiluminescence Kit (Pierce, Rockford, IL) and a charge-coupled device camera (Bio-Rad Laboratories) in a dark room, digitally scanned immune blots were analyzed using Quantity One software version 4.6.2 (Bio-Rad Laboratories). Band immune intensity ratios of CX3CL1 or TRAIL and corresponding β-actin at the same time of electrophoresis were analyzed
      • Gassmann M.
      • Grenacher B.
      • Rohde B.
      • Vogel J.
      Quantifying Western blots: pitfalls of densitometry.
      ; the ratios were the average OD values of CX3CL1 or TRAIL blot expression.

      Cell Quantification for H&E Staining

      Ten visual fields of each section were randomly chosen under a light microscope. Cell quantification was performed as described elsewhere.
      • Jung K.H.
      • Chu K.
      • Lee S.T.
      • Kim J.H.
      • Kang K.M.
      • Song E.C.
      • Kim S.J.
      • Park H.K.
      • Kim M.
      • Lee S.K.
      • Roh J.K.
      Region-specific plasticity in the epileptic rat brain: a hippocampal and extrahippocampal analysis.
      Briefly, areas or numbers of labeled cells in each section were traced and measured using an image analysis system (Image-Pro Plus Media Cybernetics, Silver Spring, MD). Slides were first examined at ×100 magnification to identify the regions and then labeled cell numbers were counted at ×400 magnification. Surviving neurons were counted in H&E-stained sections (5 μm thick). Cells with somas <3 μm were considered to be glial or necrotic cells and were excluded. The percentage of area occupied by H&E-stained neuronal nuclei was calculated for each image, and the mean value for each animal was determined by averaging values from all images taken from that animal. The mean value observed in control rats was designated as 100% of the normal cell population.

      Statistical Analysis

      Data were expressed as mean ± SD, and the analysis was performed using the Student's t-test (SPSS 11.5; SPSS Inc., Chicago, IL) between the TLE and control groups. The statistical differences in CSF and serum analyses were determined by one-way analysis of variance, combined with a post hoc Bonferroni test as a multiple comparison method, as was the statistical difference among groups of experimental animals. P < 0.05 was considered statistically significant.

      Results

      Neuronal Localization of CX3CL1

      By using double-immunofluorescence labeling, we found CX3CL1 expressed exclusively in neurons of temporal neocortex tissue from nonepileptic control and TLE patients, as shown by colocalization with the dendritic marker, MAP2 (Figure 1A). GFAP+ astrocytes were not stained (data not shown), indicating that CX3CL1 was not expressed in the astrocytes.
      Figure thumbnail gr1
      Figure 1Double-immunofluorescent labeling, IHC, and Western blot analysis for CX3CL1 in the temporal neocortex of patients with TLE and nonepileptic patients. A: Double-immunofluorescent labeling shows that the chemokine CX3CL1 (green) and MAP2 (red) are coexpressed (merged) in the temporal neocortex of an epileptic patient. White arrows, CX3CL1+/MAP2+ cells. B: IHC analysis for CX3CL1 in the temporal neocortex of humans demonstrating immunoreactive staining of CX3CL1 in the temporal neocortex of a control subject compared with strong immunoreactive staining of CX3CL1 in the temporal neocortex of a patient with TLE. Black arrows, CX3CL1+ cells. Comparison of the mean OD value (right) indicates significantly higher expression of CX3CL1 in the TLE group than in the control group. *P < 0.05. C: Western blotting analysis for CX3CL1 in the temporal neocortex of humans. Proteins from individual brain homogenates from TLE and control subjects were separated with gradient SDS-PAGE. CX3CL1 was more strongly expressed in patients with TLE than in controls. A comparison of the intensity ratio (right) indicates significantly higher expression of CX3CL1 in patients with TLE than in controls. *P < 0.05.

      Elevated CX3CL1 Protein Expression in the Temporal Neocortex of Patients with TLE

      CX3CL1 protein was expressed in the membrane and cytoplasm of neurons in the temporal neocortex of tissue from nonepileptic autopsy control and TLE patients, faint CX3CL1 staining was present in sections from the nonepileptic autopsy group, and strong immunoreactivity for CX3CL1 was observed in TLE samples (Figure 1B). No immunoreactivity was seen in negative controls in which the primary antibody had been omitted (data not shown). The mean OD value of CX3CL1 protein in the temporal neocortex tissue of patients with TLE was significantly higher than that of the nonepileptic autopsy group (0.73 ± 0.25 versus 0.30 ± 0.13; P < 0.01) (Figure 1B).
      The Western blot analysis result was in accordance with that of IHC: CX3CL1-immunoreactive bands were seen at approximately 95 kDa, and β-actin–immunoreactive bands were seen at 42 kDa. We evaluated the expression of CX3CL1 in the temporal neocortex of all TLE and nonepileptic controls. These results confirmed the slight basal level of CX3CL1 in the temporal neocortex of nonepileptic patients and its overexpression in patients with TLE (Figure 1C). Temporal neocortex samples from patients with TLE showed stronger CX3CL1 immunoreactivity when compared with normal brain tissue from nonepileptic autopsy controls (Figure 1C). The difference in the mean OD value between the TLE group and the nonepileptic control group was statistically significant (1.32 ± 0.21 versus 0.68 ± 0.11; P < 0.01) (Figure 1C).

      Elevated CX3CL1 Concentrations in Serum and CSF in Epilepsy

      We first studied the CSF concentrations of CX3CL1 in 39 patients with epilepsy, 27 control subjects with inflammatory neurological diseases, and 8 control subjects with neurosis. The level of CX3CL1 was significantly elevated in CSF samples from patients with epilepsy compared with patients with neurosis (P < 0.05) (Figure 2, A and B). In addition, the CX3CL1 level was increased in CSF samples from patients with inflammatory neurological diseases compared with both other groups (P < 0.05) (Figure 2, A and B) (epilepsy group, 0.23 ± 0.12 ng/mL; inflammatory neurological diseases group, 0.32 ± 0.15 ng/mL; neurosis group, 0.09 ± 0.07 ng/mL) (Figure 2B).
      Figure thumbnail gr2
      Figure 2CSF and serum CX3CL1 levels and the ratios of CSF CX3CL1 levels and serum CX3CL1 levels in patients with epilepsy (EP), patients with inflammatory neurological diseases, and patients with neurosis. A: CSF CX3CL1 levels in patients with EP, patients with inflammatory neurological diseases, and patients with neurosis. B: The CSF level of CX3CL1 in the EP group is increased compared with that in the neurosis group. *P < 0.05. The CSF level of CX3CL1 in the inflammatory neurological diseases group is increased compared with that in the EP group. *P < 0.05. C: Serum CX3CL1 levels in patients with EP, patients with inflammatory neurological diseases, and patients with neurosis. D: The serum level of CX3CL1 is significantly increased in the EP group compared with that in the neurosis group. *P < 0.05. The serum level of CX3CL1 in the inflammatory neurological diseases group is increased compared with that in the EP group. *P < 0.05. E: The ratios of CSF CX3CL1 over serum CX3CL1 in 21 patients with EP, 11 patients with inflammatory neurological diseases, and 8 patients with neurosis. F: The ratios of CSF to serum CX3CL1 level is not significantly different between the EP group, the inflammatory neurological diseases group, and the neurosis group. P > 0.05.
      Then, we studied the serum concentrations of CX3CL1 in 43 patients with epilepsy, 26 control subjects with inflammatory neurological diseases, and 9 control subjects with neurosis. The serum concentrations of CX3CL1 differed in patients with epilepsy, inflammatory neurological diseases, and neurosis (P < 0.01) (Figure 2, C and D). Patients with epilepsy had a higher serum concentration of CX3CL1 than patients with neurosis (0.85 ± 0.32 versus 0.41 ± 0.16 ng/mL; P < 0.01) (Figure 2D). Patients with inflammatory neurological diseases had a higher concentration of CX3CL1 than patients with epilepsy (0.85 ± 0.32 versus 0.12 ± 0.48 ng/mL; P < 0.01) (Figure 2D). The differences in CSF-CX3CL1 and serum-CX3CL1 levels in different age or sex subgroups in patients with epilepsy were not statistically significant (data not shown) (P > 0.05). Moreover, there were no significant differences in CSF or serum concentrations of CX3CL1 in epileptic patients with different disease durations (data not shown) (P > 0.05).
      The 40 CSF samples and 40 serum samples were collected from the same set of 40 patients. We analyzed the ratios of CSF concentrations of CX3CL1 to serum concentrations of CX3CL1 in the same patients. There were no significance differences in the ratios in patients with epilepsy, patients with inflammatory neurological diseases, and patients with neurosis (epilepsy group, 0.28 ± 0.14; inflammatory neurological diseases group, 0.35 ± 0.16; neurosis group, 0.25 ± 0.18; P > 0.05) (Figure 2, E and F). These results indicate that elevated serum CX3CL1 was presented in epilepsy. Blood-brain barrier (BBB) leakage is associated with seizure attacks,
      • van Vliet E.A.
      • da Costa Araujo S.
      • Redeker S.
      • van Schaik R.
      • Aronica E.
      • Gorter J.A.
      Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy.
      • Rigau V.
      • Morin M.
      • Rousset M.C.
      • de Bock F.
      • Lebrun A.
      • Coubes P.
      • Picot M.C.
      • Baldy-Moulinier M.
      • Bockaert J.
      • Crespel A.
      • Lerner-Natoli M.
      Angiogenesis is associated with blood-brain barrier permeability in temporal lobe epilepsy.
      which may contribute to blood-brain exchanges of CX3CL1. To further clarify if the increased serum CX3CL1 comes from dysfunctional brain tissues through disrupted BBB, we assessed CSF to blood albumin ratios in these three groups. Similar to those of CX3CL1, CSF to blood ratios of albumin were not significantly changed (0.0026 ± 0.0015 in epilepsy, n = 7; 0.0056 ± 0.0031 in inflammation, n = 10; and 0.0035 ± 0.0027 in neurosis, n = 4; P > 0.05; N = 21).
      To assess if the seizure frequency may have affected CX3CL1 level, we measured CSF/blood concentrations according to seizure frequencies. The seizure frequency of epileptic patients was between 0.5 and 20 times per month. We divided the epileptic patients into three subgroups: <5, 5 to 10, and 11 to 20 times per month. There was no difference of CX3CL1 in different seizure frequency subgroups between CSF and serum (P > 0.05; Figure 3, A and B).
      Figure thumbnail gr3
      Figure 3The CSF and serum CX3CL1 levels in epileptic patients with different seizure frequency subgroups. A: The CSF levels of CX3CL1 in epileptic patients with different seizure frequency subgroups. The concentrations of CX3CL1 in different seizure frequency subgroups are not significantly different: <5, 0.19 ± 0.11 ng/mL; 5 to 10, 0.25 ± 0.13 ng/mL; and 10 to 20, 0.31 ± 0.12 ng/mL. P > 0.05. B: Serum levels of CX3CL1 in epileptic patients with different seizure frequency subgroups. The concentrations of CX3CL1 in different seizure frequency subgroups are not significantly different: <5, 0.76 ± 0.31 ng/mL; 5 to 10, 0.88 ± 0.24 ng/mL; and 10 to 20, 1.02 ± 0.43 ng/mL. P > 0.05.

      Prominent CX3CL1 Expression in the Hippocampus and Adjacent Cortex in the Epileptic Rats

      To exclude the possibility that altered CX3CL1 expression may be caused by anti-epileptic drugs in patients with epilepsy, we performed an experiment in a rat model of epilepsy. CX3CL1 was expressed exclusively in neurons of the hippocampus and adjacent cortex from control and epileptic rats, as shown by colocalization with the dendritic marker, MAP2 (Figure 4A). GFAP+ astrocytes were not stained (Figure 4B). CX3CL1 was not expressed in the astrocytes.
      Figure thumbnail gr4
      Figure 4Double-immunofluorescent labeling, IHC, and Western blot analysis for CX3CL1 in the hippocampus and adjacent cortex of experimental rats. A: CX3CL1 (green) and MAP2 (red) are coexpressed (merged) in the hippocampus of a TLE rat at 72 hours after SE. White arrow, CX3CL1+/MAP2+ cell. B: CX3CL1 (green) and GFAP (red) are not coexpressed (merged). White arrow, CX3CL1+ cell; green arrow, GFAP+ cell. C: Immunoreactive staining of CX3CL1 in the hippocampus and cortex of rats. Slight immunoreactive staining of CX3CL1 in the hippocampus (CA1 field) and cortex of control rats compared with strong staining in epileptic rats at 72 hours after SE. Comparison of the mean OD value (right) of IHC staining between the control and epileptic rats at different time points after SE. *P < 0.05 versus control. Black arrows, CX3CL1+ cells. D: Western blot analysis results from control and epileptic rats. Representative Western blot analysis images showing bands of CX3CL1 and β-actin (internal control) at different time points after SE. Comparison of the mean intensity ratio (right) of immunoblotting between control and epileptic rats at each time point after SE. *P < 0.05 versus control.
      CX3CL1 was expressed in the membrane and cytoplasm of neurons of the hippocampus and adjacent cortex (Figure 4, A–C). In the epileptic groups, we observed stronger staining for CX3CL1 in neurons of the granule cell layer of the dentate gyrus, CA1 and CA3 pyramidal cell layers, and adjacent cortex (Figure 4C). IHC analysis showed that the mean OD value of each time point after SE was significantly higher than that of the control group (P < 0.01). We found an increase in CX3CL1 expression at 72 hours (298.3% of that in controls, P < 0.01), at 14 days (248.5% of that in controls, P < 0.01), and at 60 days (195.0% of that in controls, P < 0.01) (Figure 4C).
      Western blot analysis was performed to further verify the elevated CX3CL1 immunostaining observed in epileptic rat brain sections. In accordance with IHC, CX3CL1 up-regulation in epileptic tissue appeared at 6 hours after SE, reached a high level at 72 hours, and was maintained at a relatively high level until 60 days (Figure 4D). Semiquantitative densitometric analysis revealed that, in brain tissue, CX3CL1 expression of each time point after SE was significantly increased compared with the control group (P < 0.01) (Figure 4D). We found an increase in CX3CL1 expression at 72 hours (186.6% of that in controls, P < 0.01) and at 14 days (165.1% of that in controls, P < 0.01) after SE (Figure 4D).

      Neuronal Loss in the Epileptic Patients and Rats

      The H&E staining demonstrated that the anatomical sites of the samples were located in the temporal neocortex. The number of neurons was reduced in the temporal neocortex of patients with TLE compared with the number of neurons in the control tissues (P < 0.05). Neuron loss and gliosis were seen in the temporal neocortex of patients with TLE (Figure 5, A–C). The number of neurons in the temporal neocortex of patients with TLE was 53.67% in the control tissues (Figure 5D).
      Figure thumbnail gr5
      Figure 5H&E staining for the temporal neocortex of human and the hippocampus and cortex of rats. A: H&E staining for the temporal neocortex of humans. The shape and structure are relatively normal in the temporal neocortex of a control subject. The number of neurons in the control is larger than in the TLE group. Black arrow, neuronal loss; white arrow, gliosis. B: Representative H&E staining for the hippocampus and adjacent cortex of rats. The numbers of neurons in controls are greater than those at 7 and 60 days. Black arrows, areas of massive neuron loss in the hippocampus and adjacent cortex of epileptic rats; white arrows, gliosis in the hippocampus and adjacent cortex of epileptic rats. C: The percentage of surviving neurons in the TLE group compared with the control group. The number of neurons in the TLE group is significantly lower than in the control group. *P < 0.05. D: The percentages of surviving neurons in the 72-hour group, the 7-day group, and the 60-day group, compared with the control group. The number of neurons in epileptic groups is significantly lower than in the control group. *P < 0.05.
      In a rat model of epilepsy, we counted the number of surviving neurons in the hippocampus and adjacent cortex of control rats and rats after SE at representative time points: 72 hours, 7 days, and 60 days. H&E staining demonstrated that the number of neurons was reduced in the hippocampus and adjacent cortex of the epileptic rats compared with the number of neurons in control rats. Neuron loss and gliosis were seen in the hippocampus and adjacent cortex of the epileptic rats. We found a decrease in the number of surviving neurons at 72 hours (89.34% of that in controls, P < 0.01), 7 days (83.26% of that in controls, P < 0.01), and 60 days (61.75% of that in controls, P < 0.01) after SE (Figure 5).

      Prominent TRAIL Expression in Epileptic Patients and Rats

      TRAIL was expressed in the membrane and cytoplasm of cells of the temporal neocortex of patients (Figure 6A). A Western blot analysis study showed that TRAIL was significantly increased in epileptic patients (Figure 6B). TRAIL-immunoreactive bands were seen at approximately 34 kDa, and β-actin–immunoreactive bands were seen at 42 kDa. We evaluated the expression of TRAIL in the temporal neocortex of patients with TLE and nonepileptic controls. Temporal neocortex samples from patients with TLE showed stronger TRAIL immunoreactivity when compared with normal brain tissue from nonepileptic autopsy controls (Figure 6B). The difference in mean OD values between the TLE and nonepileptic control groups was statistically significant (1.23 ± 0.52 versus 0.47 ± 0.15; P < 0.01) (Figure 6B).
      Figure thumbnail gr6
      Figure 6IHC and Western blot analysis for TRAIL in the temporal neocortex of patients with TLE and nonepileptic patients. A: IHC analysis for TRAIL in the temporal neocortex of humans. Arrows, TRAIL+ cells. Slight immunoreactive staining of TRAIL is shown in the temporal neocortex of a control subject compared with strong staining in a patient with TLE. B: Western blot analysis for TRAIL in the temporal neocortex of humans. Proteins from individual brain homogenates from TLE and control subjects were separated with gradient SDS-PAGE. Immunoreactive stainings of TRAIL in patients with TLE are stronger than those in controls. A comparison of the mean intensity ratio (right) indicates significantly higher expression of TRAIL in patients with TLE than in controls. *P < 0.05.
      TRAIL was expressed in the membrane and cytoplasm of neurons and astrocytes in the hippocampus and adjacent cortex of rats (Figure 7, A and B). We found TRAIL expressed in neurons and astrocytes in epileptic rats, as shown by colocalization with the dendritic marker, MAP2 (Figure 7A), or the astrocyte marker, GFAP (Figure 7A), indicating that TRAIL was expressed in both neurons and astrocytes. Colocalization of TRAIL with FJB-stained cells suggests that TRAIL is included in injured neurons (Figure 7A).
      Figure thumbnail gr7
      Figure 7Double-immunofluorescent labeling, IHC, and Western blot analysis for CX3CL1 in the hippocampus and adjacent cortex of experimental rats. A: TRAIL (red) and MAP2 (green) are coexpressed (merged) in neurons of cortex from a TLE rat at 60 days after SE. TRAIL (red) and GFAP (green) are coexpressed (merged) in astrocytes of cortex from a TLE rat at 30 days after SE. TRAIL+ cells (red) also express FJB (green), indicating degeneration. White arrows, TRAIL+/MAP2+ cell (top panels), TRAIL+/GFAP+ cell (middle panels), TRAIL+/FJB+ cell (bottom panels). B: Immunoreactive staining of TRAIL in the hippocampus and cortex of rats (6 hours). Slight immunoreactive staining of TRAIL is shown in the cortex of 6-hour rats (60 days) compared with strong staining in the hippocampus (CA1 field) and cortex of epileptic rats at 60 days after SE. Black arrows, TRAIL+ cells. C: Western blot results from control and epileptic rats. Left panel: Representative Western blot analysis images of TRAIL and β-actin (internal control) at different time points after SE. A comparison of the intensity ratio (right) of immunoblots between control and epileptic rats at each time point after SE. *P < 0.05 versus control.
      Western blot analysis was performed to evaluate the expression of TRAIL in the hippocampus and adjacent cortex of epileptic rats. TRAIL up-regulation in epileptic tissue appeared at 6 hours after SE, gradually increased along with the time after seizures, and reached a high level at 60 days (Figure 7B). Semiquantitative densitometric analysis revealed that, in brain tissue, TRAIL expression of each time point after SE was significantly increased compared with the control group (P < 0.05) (Figure 7C).

      Discussion

      The major findings of this study are as follows: i) CX3CL1 is significantly up-regulated in neurons from patients with TLE and lithium chloride–pilocarpine–induced rats; ii) CSF and serum concentrations of CX3CL1 are significantly increased in patients with epilepsy; iii) the neuronal loss is apparent in epileptic patients and rats; and iv) consistent with neuronal death, TRAIL is significantly increased in both patients and the animal model.
      Although the underlying mechanisms of epileptogenesis are still unknown, inflammation mechanisms, such as pro-inflammatory cytokines, play a critical role in the pathogenesis of epilepsy.
      • Vezzani A.
      • Granata T.
      Brain inflammation in epilepsy: experimental and clinical evidence.
      • Fabene P.F.
      • Bramanti P.
      • Constantin G.
      The emerging role for chemokines in epilepsy.
      Chemokines can modulate neuronal activity, as follows: i) modulation of voltage-dependent channels (sodium, potassium, and calcium); ii) activation of the G-protein–activated inward rectifier potassium current; and iii) increase of neurotransmitter release (GABA, glutamate, and dopamine), often through calcium-dependent mechanisms.
      • Lauro C.
      • Di Angelantonio S.
      • Cipriani R.
      • Sobrero F.
      • Antonilli L.
      • Brusadin V.
      • Ragozzino D.
      • Limatola C.
      Activity of adenosine receptors type 1 is required for CX3CL1-mediated neuroprotection and neuromodulation in hippocampal neurons.
      In the present study, CX3CL1 expression was up-regulated in patients and an animal model. Epilepsy involves molecular factors, cellular alterations, and neuronal network reorganization.
      • Rakhade S.N.
      • Jensen F.E.
      Epileptogenesis in the immature brain: emerging mechanisms.
      • Wong M.
      Modulation of dendritic spines in epilepsy: cellular mechanisms and functional implications.
      Increased expression of CX3CL1 may control neuronal excitability by modulating glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) currents,
      • Fabene P.F.
      • Bramanti P.
      • Constantin G.
      The emerging role for chemokines in epilepsy.
      • Lauro C.
      • Di Angelantonio S.
      • Cipriani R.
      • Sobrero F.
      • Antonilli L.
      • Brusadin V.
      • Ragozzino D.
      • Limatola C.
      Activity of adenosine receptors type 1 is required for CX3CL1-mediated neuroprotection and neuromodulation in hippocampal neurons.
      although its exact role in the epilepsy phenotype remains unclear.
      Consistent with the finding in the brain tissue, our research showed that levels of CX3CL1 in CSF and serum of patients with epilepsy were significantly higher than those in patients with neurosis. However, we did not find an increased CSF/serum ratio in patients versus controls. The increased serum CX3CL1 may be attributed to BBB leakage in epilepsy. BBB disruption and angiogenesis are associated with TLE and an experimental animal model.
      • Rigau V.
      • Morin M.
      • Rousset M.C.
      • de Bock F.
      • Lebrun A.
      • Coubes P.
      • Picot M.C.
      • Baldy-Moulinier M.
      • Bockaert J.
      • Crespel A.
      • Lerner-Natoli M.
      Angiogenesis is associated with blood-brain barrier permeability in temporal lobe epilepsy.
      Evidence has also shown that BBB leakage increases seizure frequency.
      • van Vliet E.A.
      • da Costa Araujo S.
      • Redeker S.
      • van Schaik R.
      • Aronica E.
      • Gorter J.A.
      Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy.
      An impaired tight junction may allow circulating IgG to be accumulated in neurons through a compromised BBB, which may contribute to epileptogenesis. Conversely, increased expression of brain fractalkine gains access to the blood flow and results in an elevated serum level, although direct evidence is lacking. Therefore, we assume that augmentation of fractalkine in both CSF and serum resulted in relatively unchanged CSF/serum ratios. The less CX3CL1 in epilepsy than in inflammatory central nervous system diseases suggests that inflammatory processes in epilepsy may not be a prominent feature (Figure 2).
      • Kastenbauer S.
      • Koedel U.
      • Wick M.
      • Kieseier B.C.
      • Hartung H.P.
      • Pfister H.W.
      CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system.
      In this study, the number of neurons was reduced after epilepsy, which is consistent with previous studies.
      • Li J.M.
      • Wang X.F.
      • Xi Z.Q.
      • Gong Y.
      • Liu F.Y.
      • Sun J.J.
      • Wu Y.
      • Luan G.M.
      • Wang Y.P.
      • Li Y.L.
      • Zhang J.G.
      • Lu Y.
      • Li H.W.
      Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy.
      • Wang L.
      • Liu G.
      • He M.
      • Shen L.
      • Shen D.
      • Lu Y.
      • Wang X.
      Increased insulin receptor expression in anterior temporal neocortex of patients with intractable epilepsy.
      • Xi Z.Q.
      • Wang X.F.
      • He R.Q.
      • Li M.W.
      • Liu X.Z.
      • Wang L.Y.
      • Zhu X.
      • Xiao F.
      • Sun J.J.
      • Li J.M.
      • Gong Y.
      • Guan L.F.
      Extracellular signal-regulated protein kinase in human intractable epilepsy.
      Evidence indicates that cell death and inflammation parallel the time course of hippocampal remodeling after seizure. In support of this, intracerebroventricular infusions of recombinant rat CX3CL1 aggravated SE-induced neuronal damage.
      • Yeo S.I.
      • Kim J.E.
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      Both necrosis and apoptosis may contribute to neuronal loss, depending on seizure activity and cell vulnerability. Although necrosis is prominent in SE, a condition in which repetitive seizure activity lasts for tens to hundreds of minutes,
      • Fujikawa D.G.
      Prolonged seizures and cellular injury: understanding the connection.
      apoptosis is associated with both SE and multiple brief seizures.
      • McNamara J.O.
      Emerging insights into the genesis of epilepsy.
      • Henshall D.C.
      Apoptosis signalling pathways in seizure-induced neuronal death and epilepsy.
      On the other hand, different neuron populations may have varied vulnerability. Prolonged afferent stimulation leads to necrosis in hilar neurons and pyramidal cells in the hippocampus, but the same stimulation causes apoptosislike morphological features in dentate granule cells.
      • Sloviter R.S.
      • Dean E.
      • Sollas A.L.
      • Goodman J.H.
      Apoptosis and necrosis induced in different hippocampal neuron populations by repetitive perforant path stimulation in the rat.
      Up-regulation of inflammation mediators IL-1, tumor necrosis factor, and IL-6 after seizures contribute to neuronal death.
      • Vezzani A.
      • French J.
      • Bartfai T.
      • Baram T.Z.
      The role of inflammation in epilepsy.
      • Jankowsky J.L.
      • Patterson P.H.
      The role of cytokines and growth factors in seizures and their sequelae.
      However, whether TRAIL is also deregulated in epilepsy is relatively unclear. Our study showed that TRAIL was significantly increased in patients and an animal model. Colocalization of TRAIL with FJB-stained cells suggests that TRAIL is included in injured neurons (Figure 7A). There was a trend that expression of TRAIL was increased along with the time after seizures, which was consistent with that of fractalkine. These results suggest that TRAIL is involved in cell death in epilepsy. Surprisingly, fractalkine has been protective. Incubation of fractalkine in hippocampal neurons promotes survival.
      • Meucci O.
      • Fatatis A.
      • Simen A.A.
      • Miller R.J.
      Expression of CX3CR1 chemokine receptors on neurons and their role in neuronal survival.
      Bath application of fractalkine in hippocampal slices depresses excitatory postsynaptic current and AMPA-type glutamate receptor current, whereas fractalkine knockout mice result in increased currents.
      • Ragozzino D.
      • Di Angelantonio S.
      • Trettel F.
      • Bertollini C.
      • Maggi L.
      • Gross C.
      • Charo I.F.
      • Limatola C.
      • Eusebi F.
      Chemokine fractalkine/CX3CL1 negatively modulates active glutamatergic synapses in rat hippocampal neurons.
      Then, how may deregulated fractalkine cause cell death? One possibility is that epilepsy is accompanied by prominent BBB damage,
      • van Vliet E.A.
      • da Costa Araujo S.
      • Redeker S.
      • van Schaik R.
      • Aronica E.
      • Gorter J.A.
      Blood-brain barrier leakage may lead to progression of temporal lobe epilepsy.
      • Rigau V.
      • Morin M.
      • Rousset M.C.
      • de Bock F.
      • Lebrun A.
      • Coubes P.
      • Picot M.C.
      • Baldy-Moulinier M.
      • Bockaert J.
      • Crespel A.
      • Lerner-Natoli M.
      Angiogenesis is associated with blood-brain barrier permeability in temporal lobe epilepsy.
      which allows circulating mononuclear phagocytes to be attracted by fractalkine, because it is clear that macrophages express fractalkine receptor CXLCR1
      • Oh D.J.
      • Dursun B.
      • He Z.
      • Lu L.
      • Hoke T.S.
      • Ljubanovic D.
      • Faubel S.
      • Edelstein C.L.
      Fractalkine receptor (CX3CR1) inhibition is protective against ischemic acute renal failure in mice.
      and TRAIL.
      • Huang Y.
      • Erdmann N.
      • Peng H.
      • Zhao Y.
      • Zheng J.
      The role of TNF related apoptosis-inducing ligand in neurodegenerative diseases.
      The latter, in turn, mediates inflammation cascades and cell death in the affected brain region
      • Huang Y.
      • Erdmann N.
      • Peng H.
      • Zhao Y.
      • Zheng J.
      The role of TNF related apoptosis-inducing ligand in neurodegenerative diseases.
      (Figure 7A). Evidence has indicated that circulating macrophages play an important role in CXLCR1-mediated ischemic renal failure.
      • Oh D.J.
      • Dursun B.
      • He Z.
      • Lu L.
      • Hoke T.S.
      • Ljubanovic D.
      • Faubel S.
      • Edelstein C.L.
      Fractalkine receptor (CX3CR1) inhibition is protective against ischemic acute renal failure in mice.
      Another possibility is that brain macrophages and microglia also express TRAIL (Figure 7A48), deregulation of which may also lead to neuronal death.
      In conclusion, our study demonstrates, for the first time to our knowledge, the increased expression of CX3CL1 in the temporal neocortex, CSF, and serum of patients with epilepsy, which was associated with neuronal loss and increased TRAIL expression. These findings also support that CXCL13 may serve as a biomarker for epilepsy. However, the conclusion in this study is purely hypothetical, because results in this study are descriptive and did not provide evidence showing that CX3CL1 contributes to neuronal loss. Although altered expression of CX3CL1 developed after epilepsy, whether CX3CL1 also causes cell death by TRAIL or is involved in the development of epilepsy remains to be further clarified.
      This study also had limitations. In this study, most patients who provided brain specimens were unwilling to provide blood and CSF samples. All blood/CSF samples were from patients in the Department of Neurology (The First Affiliated Hospital, Chongqing Medical University) (another group of epileptic patients). Thus, this study did not directly link brain CX3CL1 with CFS/blood CX3CL1 from the same patients undergoing surgery. Although clinical features, including seizure type and frequencies, appear to be similar (Table 1, Table 3) in these two groups, the underlying pathophysiological mechanisms may not necessarily be the same, requiring a better-designed experiment in the future.

      Acknowledgments

      We sincerely thank the subjects and their families for their participation in this study.

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