Brain-derived neurotrophic factor expression is higher in brain tissue from patients with refractory epilepsy than in normal controls＊☆

Department of Neurology, First Hospital of Jilin University, Changchun 130021, Jilin Province, China

lNTRODUCTlON

Brain-derived neurotrophic factor (BDNF)can promote neural development and benefit synaptic reconstruction[1-2].Dual effects of BDNF have been observed in epilepsy[3].For instance, increased BDNF levels may be epileptogenic[4], but may also suppress seizures, protect against brain injury and inhibit neuronal discharge postictally[5].In the mature brain, BDNF binding to receptors can enhance neuronal excitability[6-7].Upregulated BDNF expression has been detected in several animal models of epilepsy and in brain tissue from epileptic patients[6-7].Moreover,BDNF overexpression can induce epileptic seizures[8]and exacerbate epilepsy-induced brain injury[9-10].However, some studies indicate that BDNF can promote functional reconstruction following nerve injury repair[11-13].

Unsainet al[9]demonstrated that BDNF expression was reduced following seizures,causing inhibition of postictal neuronal injury,whereas the damage was aggravated if BDNF expression was increased.Results of our previous studies support that BDNF directly participates in seizure incidence and progression as well as neuronal injury[14-16].Approximately 20-25% of epileptic patients are refractory to anti-epileptic drugs and suffer intractable epilepsy[17].Takahashiet al[18]reported that BDNF expression in the temporal cortex of patients with refractory epilepsy was 2.6 times higher than normal.The present study investigated BDNF expression and ultrastructural changes in resected seizure foci of patients with refractory epilepsy, to further understand the role of BDNF in refractory epilepsy.

RESULTS

Quantitative analysis of participants

A total of 30 patients with refractory epilepsy were selected, and six withdrew from the follow-up, giving a total of 24 participants in the case group.Six healthy people were selected as controls.

Baseline data of participants

The case group comprised 12 males and 12 females, aged 28.3±11.1 years (range 17-58 years), with an average disease duration of 8.3±7.8 years (range 2-29 years).The seizure frequency was 4-40 times per month in the past 6 months.The baseline data of the 24 patients are presented in Table 1 and in supplementary Figures 1-8 online.The control group included four males and two females, aged 36.5±16.8 years (range 17-55 years).

Pathological changes in the seizure focus

Under the light microscope, the cerebral cortical layers were disordered, with an uneven distribution of neurons and the presence of immature and degenerated neurons.

Table 1 Baseline data of 24 patients with refractory epilepsy

Nuclear vacuoles, reduced cytoplasm, glial cell proliferation, neural cell tropism, and small-vessel hyperplasia and hyperemia could be seen.In addition,scattered lymphocytes and plasma cell infiltration were observed, with lymphocyte infiltration surrounding the vessels (Figure 1).

Figure 1 Pathology of tissue from the seizure foci of epileptic patients and at corresponding sites of non-epileptic controls (hematoxylin-eosin staining, × 400).

Under the electron microscope, epileptic tissue presented neural cells degeneration and necrosis,pyknosis, aberrant nuclear membrane structures,nuclear vacuolization, mitochondrial swelling, cristae breakage, increased intracytoplasm density, swollen astrocytes, and cytoplasmic vacuoles.In addition,chromatin margination and nuclear membrane breakage were occasionally observed (Figure 2).

Figure 2 Cellular ultrastructure of the epileptic seizure focus.

BDNF expression in the seizure focus

Transmission electron microscope observations revealed cytoplasmic BDNF protein expression in neurons in both epileptic tissue and normal controls.The number of BDNF-positive particles was 98.16±11.09/20 000-fold visual field in the case group and 30.13±9.81/20 000-fold visual field in the control group (P＜0.01; Figure 3).

Figure 3 Brain-derived neurotrophic factor expression in the seizure foci of epileptic patients and at corresponding sites of non-epileptic controls (colloidal gold staining,× 20 000).

Immunohistochemistry showed that BDNF was expressed in neurons and glial cells in both groups.The number of BDNF positive cells was 97.42±7.09/400-fold visual field in the case group and 26.98±6.11/400-fold visual field in the control group (light microscope;P＜0.01; Figure 4).

DlSCUSSlON

BDNF can inhibit epileptic seizures, promote regeneration of injured neurons, participate in neuronal remodeling after seizures[19], and play a role in synaptic function[20].However, some studies suggest that BDNF can enhance neuronal excitability in the hippocampus,resulting in recurrent seizures[21-23].

Most studies on the role of BDNF in epilepsy have focused on animal experiments.The present study selected patients with refractory epilepsy and quantified BDNF expression changes in seizure foci.This approach is more clinically significant than animal studies for further understanding the role of BDNF in epilepsy.However, because the samples in the present study were from humans, the epilepsy process was not controllable and the time point of sample collection was not uniform.

Figure 4 Brain-derived neurotrophic factor expression in the seizure foci of epileptic patients and at corresponding sites of non-epileptic controls (immunohistochemical staining, × 400).Brown-yellow stained cell membranes were regarded as positive.

Our findings indicate that high BDNF expression is linked to epilepsy, consistent with our previous animal studies[14-16].In these studies, we used the amygdala kindling rat model of temporal lobe epilepsy to investigate brain damage and BDNF expression after seizures.The results showed neuronal degeneration, cell swelling, and necrosis in the temporal cortex and hippocampus, and the changes were most severe seven days after the start of kindling[14-16].The hippocampus showed more damage than the temporal cortex, and the number of BDNF-positive neurons was correlated with kindling times and degree of neuronal injury, indicating that BDNF expression increases in the brain in response to epileptic seizures.This may be an endogenous protective reaction to prevent cell death.Further studies are needed to investigate BDNF expression in refractory epilepsy.

SUBJECTS AND METHODS

Design

A case-control study of neuropathology.

Time and setting

This study was performed at the Department of Pathology, School of Basic Medicine and Department of Neurology, First Hospital of Jilin University, China in 2011.

Subjects

The subjects were 24 patients with refractory epilepsy that underwent resection of seizure foci at the Epilepsy Center, First Hospital of Jilin University, between May 2010 and April 2011.They included 12 males and 12 females, aged 28.3±11.1 years (range 17-58 years),with a disease duration of 8.3±7.8 years (range 2-29 years).

Inclusion criteria: The clinical diagnosis was made according to the Proposal for revised clinical and electroencephalographic classification of epileptic seizures and Proposal for revised classification of epilepsies and epileptic syndromes (The Commission on Classification and Terminology of the International League Against Epilepsy)[24-25].Subjects were examined by ordinary electroencephalogram (EEG), long-term video-EEG and cortical EEG using an EEG-9000 long-term video EEG monitor (Nihon Kohden Corporation, Japan).The international 10-20 system was used for electrode placement, and sphenoidal electrode recordings were made from bilateral temporal regions.During the EEG examination, the patients underwent a routine eye open-close test,hyperventilation test, and flash stimulation test.The seizures were recorded on video.The average potential reference was used in EEG reviews.In addition, head MRI was performed (1.5 or 3.0 T).Only patients with uncontrollable seizures (＞ 4 seizures/month, ＞ 2 years),despite treatment with two or more anti-epileptic drugs(phenytoin, carbamazepine, valproic acid or phenobarbital) were included.Informed consent was obtained from all participants.

Six patients with craniocerebral trauma were selected from Department of Neurosurgery, Jilin University, as a control group.They were free of epilepsy and other central nervous system diseases.They comprised 4 males and 2 females, aged 36.8±16.3 years (range 19-55 years).

Methods

Harvest of brain tissues

Temporal lobe tissue was harvested from both groups,and fixed with 4% paraformaldehyde, dehydrated with gradient alcohol, cleared with xylene, paraffin-embedded,sectioned (3 μm), and stained with hematoxylin-eosin[26].The sections were observed using a light microscope(Olympus, Tokyo, Japan).

Transmission electron microscopy observation of ultrastructure and BDNF expression

Tissues were immersed in 4% glutaraldehyde, fixed in 1% osmic acid, dehydrated with gradient alcohol,embedded in Epon812 epoxy resin, sectioned into semi-thin slices (60-70 nm), and stained with uranyl acetate and lead citrate.The sections were incubated with colloidal gold-labeled goat anti-rabbit IgG (1: 100;Beijing Biosynthesis Biotechnology, Beijing, China).Five sections were selected from each subject.Ultrastructural changes were investigated using a JEM-1000SX transmission electron microscope (Summit Medical Co.,Tokyo, Japan) and the quantity of BDNF-positive particles was calculated (× 20 000).

BDNF immunohistochemistry

The paraffin sections were dewaxed, hydrated, and incubated with 0.1% trypsin at 37°C for 10 minutes,followed by peroxidase at room temperature for 10 minutes.The sections were incubated with rabbit anti-BDNF polyclonal antibody (1: 1 000; Wuhan Boster,China) for 2 hours at room temperature, and overnight at 4°C, followed by biotinylated goat anti-rabbit IgG (1: 200;Fuzhou Maxim, China) at room temperature for 2 hours.The sections were colorized with a solution containing 0.05% diaminobenzidine (Boster) and 0.01% H2O2.Three 3-5 minutes PBS (pH 7.4) washes were performed between each step.Cell morphology was observed under a light microscope (Olympus).The sections were dehydrated with gradient alcohol after 5-10 minutes, counterstained with hematoxylin, and mounted with neutral gum.PBS was used as a negative control[27-28].Five sections from each subject were selected, and five fields of view were randomly selected(× 400) to quantify the number of BDNF-positive cells.The average value was calculated.

Statistical analysis

Data were analyzed using Excel 2000 (Microsoft,Chicago, IL, USA) and expressed as mean±SD.Intragroup differences were compared utilizing pairedt-tests.The significance level wasα=0.05.

Author contributions:Yudan Lv participated in data analysis.Jiqing Qiu provided brain tissue samples.Zan Wang performed statistical analysis.Li Cui collected data from patients and provided technical support.Weihong Lin was in charge of funds,conceived and designed this study, and wrote the manuscript.Hongmei Meng provided and integrated experimental data.

Conflicts of interest:None declared.

Funding:This study was supported by the “211 Project”Foundation of Jilin University (Diagnosis and treatment of major nervous system disease), No.450101120008.

Ethical approval:This study was approved by the Ethics Committee of First Hospital, Jilin University, China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol.6, No.29, 2011 item after selecting the “NRR Current Issue” button on the page.

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