Cell Mol Neurobiol DOI 10.1007/s10571-015-0181-y
ORIGINAL RESEARCH
Altered Expression of Intersectin1-L in Patients with Refractory Epilepsy and in Experimental Epileptic Rats Xiaoyan Yang1 • Xin Xu1 • Yujiao Zhang1 • Shasha Wang2 • Minghui Li2 Xuefeng Wang1
•
Received: 15 December 2014 / Accepted: 11 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Epilepsy is a common neurological disorder. Because its underlying mechanisms remain incompletely understood, current treatments are not adequate for all epilepsy patients, and some patients progress to refractory epilepsy. Under physiological conditions, excitatory and inhibitory neurons function in a dynamic balance. Epilepsy develops when this balance is disrupted. Intersectin1-L is a major scaffold protein in the central nervous system that contains multiple functional domains, and it is the long form of intersectin1. Recent studies have shown that intersectin1-L plays an important role in the process of neurotransmitter release. In this study, we investigated the expression pattern and distribution of intersectin1-L in patients with refractory epilepsy, in a rat model of pilocarpine-induced epilepsy, and in a rat model of amygdalakindled epilepsy by immunohistochemistry, immunofluorescence, and Western blotting. The purpose of this study was to explore the relationship between epilepsy and intersectin1-L. The results showed that the intersectin1-L protein was primarily expressed in neurons in brain tissue. Its expression was remarkably increased in patients with refractory epilepsy and in epilepsy model rats. These results suggest that the abnormal expression of the intersectin1-L protein in epileptic brain tissue may play an important role in epilepsy, especially refractory epilepsy. Xiaoyan Yang and Xin Xu have contributed equally to this work. & Xuefeng Wang [email protected] 1
Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Chongqing 400016, China
2
Chongqing Medical University, No.1 Yixueyuan Road, Chongqing 400016, China
Keywords
Intersectin1-L Patient Epilepsy Rat model
Introduction Epilepsy is a common disease of the nervous system with a high incidence. Approximately 50 million people suffer from epilepsy worldwide (Dulac and Milh 2012), and onethird of epilepsy patients develop refractory epilepsy after unsuccessful drug treatment. Repeated seizures may cause neuronal damage, resulting in irreversible brain damage and poor prognosis (Towne 2007). Epilepsy causes substantial economic and psychological burdens on patients and their families. The pathology of epilepsy is based on the abnormal synchronization of neuronal discharges (Leach and Abassi 2013). However, the mechanism underlying refractory epilepsy remains to be fully elucidated. Under physiological conditions, neuronal excitation and inhibition are maintained in dynamic equilibrium. This balance can be disrupted by abnormal neurotransmitter release. Altered neuronal excitability causes abnormal discharges, which may lead to the development of epilepsy. Intersectin is a scaffold protein that contains multiple functional domains (O’Bryan et al. 2001). The human intersectin gene encodes two intersectin proteins, intersectin1 and intersectin2. Each protein has two isoforms, referred to as intersectin-L and intersectin-S (Kropyvko et al. 2010; Yu et al. 2008), which are expressed in tissuespecific manners. Intersectin1-L is primarily expressed in neurons of the central nervous system (Ma et al. 2003) and plays an important role in synaptic neurotransmitter release (Ma et al. 2003; Sakaba et al. 2013). Intersectin1-S is primarily expressed in glial cells and plays important roles in cell apoptosis and cytoskeleton reorganization (Ma et al. 2010, 2011).
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Temporal lobe epilepsy (TLE) is the most common type of refractory epilepsy (Beleza 2009; Kwan and Brodie 2000). Considering the localization of intersectin1-L expression and its regulatory function in neurotransmitter release, we hypothesized that its expression in the brain tissue of refractory epilepsy patients and TLE animal models may be altered and that such alterations may be involved in the development of refractory epilepsy. To test this hypothesis, we performed immunofluorescence, immunohistochemistry, and Western blot analyses to detect the expression of intersectin1-L in the brain tissues of patients with refractory epilepsy and of animal models of TLE.
Materials and Methods All patients enrolled in this study provided written informed consent. Human brain tissue samples were obtained from Xuanwu Hospital, Capital Medical University, Tiantan Hospital, the Third Military Medical University, Xinqiao Hospital and the First Affiliated Hospital of Chongqing Medical University. This study was approved by the Ethics Committee of Chongqing Medical University. The entire study protocol was in compliance with the Declaration of Helsinki of the World Medical Association guidelines. Human Brain Tissue Human Brain Tissue and Clinical Data We constructed a brain tissue library comprising 223 brain tissue samples from patients with refractory TLE and 60 brain tissue samples from a control group of patients. The epilepsy patients exhibited typical clinical characteristics and changes in their EEG, and they met the 2001 International League Against Epilepsy diagnostic criteria for epilepsy. In these patients, treatment with the maximum doses of three types of antiepileptic drugs was unsuccessful, and surgery was elected. Preoperative evaluation included a detailed medical history, neurological examination, radiographic examination, and routine preoperative examination. The epileptic focus was localized via video EEG or 24-h ambulatory EEG prior to surgery, and surgery was performed during sphenoidal electrode monitoring and intraoperative electrocorticography. After excision of the epileptic focus, EEG of the perilesional tissue was performed to verify the complete excision of the lesion. The pathological assessment of the excised lesion revealed neuronal degeneration or gliosis, which is consistent with the pathological manifestations of epilepsy. The brain tissue of the control group was obtained from a
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resected area that was excised during decompression after a traumatic brain injury. The results of the pathological assessment of the brain tissue from the control group were normal. The clinical records of the control patients indicated that they had no history of epilepsy or the use of antiepileptic medication. For the present study, we randomly selected 22 samples from the epilepsy patient group and 10 samples from the control group from the brain tissue library. The clinical data are shown in Tables 1 and 2. Human Brain Tissue Preparation The collected specimens were divided into three groups. One group of specimens was stored in liquid nitrogen for Western blotting to detect the protein expression of intersectin1-L. The remaining two groups of specimens were immediately fixed with 4 % phosphate-buffered formalin for 48 h. After fixation, one of these groups of specimens was frozen and sectioned at a thickness of 10 lm for immunofluorescence to determine the localization of intersectin1-L expression. The remaining group was sectioned in paraffin at a thickness of 5 lm for immunohistochemistry to detect the expression of intersectin1-L. Brain tissue from Animal Models of Epilepsy Animal Selection and Breeding Healthy adult Sprague–Dawley rats (7–8 weeks old, 200–240 g) provided by the Chongqing Medical University Animal Center were used for this experiment. The animals were housed at 24–26 °C with a relative humidity of 50–60 % under an artificial 12 h light–dark cycle to maintain their circadian rhythm and were provided with free access to food and water. Pilocarpine-Induced Animal Model of Epilepsy Sixteen healthy adult rats were randomly assigned to a control group or epilepsy group (n = 8). Each rat in the epilepsy group was administered 127 mg/kg lithium chloride (Sigma, USA) and after 12 h, received 50 mg/kg pilocarpine (Sigma, USA) via intraperitoneal injection. Because status epilepticus (SE) may not always appear after the first injection of pilocarpine, the treatment was repeatedly administered until the onset of SE. In most cases, pilocarpine administration was repeated 5 times, with 30 min intervals between each injection. After the onset of SE, seizures were rated from 1 to 5 according to Racine’s scale. Animals receiving a score of 4 or higher were included in follow-up studies. For cases in which SE exceeded 60 min, diazepam (10 mg/kg) (Sigma, USA) was administered to terminate the seizures. After the
Cell Mol Neurobiol Table 1 Clinical characteristics of intractable epilepsy patients Patient
Sex (M/F)
Age (years)
Course (years)
AEDs before surgery
Source of organization
Pathologic result
1
M
15
7
LTG/TPM/CBZ
RTN
NL/G
2
F
32
16
VPA/PB/CBZ
LTN
NL/G
3
M
18
4
CBZ/VPA/PHT
RTN
NL/G
4
F
22
6
VPA/CBZ/PB
RTN
NL/G
5
M
16
3
VPA/TPM/CBZ
RTN
G
6
F
35
18
VPA/PB/CBZ/LEV
LTN
NL/G
7
M
17
5
VPA/CBZ//TPM
LTN
NL/G
8
M
23
5
CBZ/VPA/PHT/PB
LTN
NL/G
9
M
26
12
PHT/VPA/PB/TPM
LTN
NL/G
10
F
16
6
CBZ/LEV/TPM/LTG
RTN
NL/G
11 12
M M
25 16
8 4
VPACBZ/TPM VPA/OXC/TPM/PB
RTN LTN
NL/G G
13
M
18
3
VPA/CBZ/TP
RTN
G
14
F
21
7
CBZ/PB/LTG/LEV
RTN
NL/G
15
F
28
11
CBZ/PHT/PB/LTG
LTN
NL/G
16
M
24
12
CBZ/PHT/VPA/PB
LTN
NL/G
17
M
19
8
VPA/CBZ/PB
RTN
NL/G
18
F
20
5
CBZ/LTG/LEV
RTN
NL/G
19
M
39
15
CBZ/VPA/PHT/PB
RTN
NL/G
20
M
36
12
VPA/PHT/CBZ/PB
LTN
NL/G
21
M
23
8
VPA/CBZ/PHT
LTN
NL/G
22
F
25
12
CBZ/PB/LTG/LEV
LTN
NL/G
F female, M male, AEDs antiepilepti drugs, LTG lamotrigine, TPM topiramate, CBZ carbamazepine, PB phenobarbital, VPA valproic acid, LEV levetiracetam, OXC oxcarbazepine, RTN right temporal neocortex, LTN left temporal neocortex, NL neuronal necrosis, G gliosis
Table 2 Clinical characteristics of control group
Patient
Sex
Age (years)
Disease diagnosis
Source of organization
Pathologic result
1
M
25
Brain trauma
LTN
Normal
2
M
19
Brain trauma
LTN
Normal
3
M
36
Brain trauma
RTN
Normal
4
M
14
Brain trauma
RTN
Normal
5 6
F F
23 21
Brain trauma Brain trauma
LTN RTN
Normal Normal
7
M
16
Brain trauma
LTN
Normal
8
F
28
Brain trauma
RTN
Normal
9
M
19
Brain trauma
RTN
Normal
10
F
20
Brain trauma
LTN
Normal
F female, M male, RTN right temporal neocortex, LTN light temporal neocortex
termination of SE, we observed whether spontaneous seizures occurred in the rats during the chronic phase. Rats exhibiting spontaneous seizures were included in the epilepsy group. The control group of rats was injected with normal saline instead of lithium chloride and pilocarpine and underwent an identical surgical procedure.
Amygdala-Kindled Epilepsy Animal Model Sixteen healthy adult rats were randomly assigned to a control group or epilepsy group (n = 8), and 3.5 % chloral hydrate was administered via intraperitoneal injection at 1 ml/100 g by weight. The rats were anesthetized and fixed to a stereotaxic frame. The skull was fully exposed, and the
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position of the amygdala was located (2 mm posterior and 5 mm lateral to the bregma at a depth of 8 mm). After drilling through the skull, an electrode was implanted and fixed using dental cement. The postoperative rats were reared under the same conditions as the preoperative rats. Stimulation was initiated at 8 days after the surgical procedure (stimulation parameters: train stimulation, square wave, and wave width of -1 ms, and frequency of -50 Hz). The rats were stimulated once daily for 10 consecutive days. The model was considered to be successful when the rats exhibited seizures (according to Racine’s scale, Cstage 4) (Madsen et al. 2006; Pitka¨nen et al. 2005). The control group underwent stereotactic surgery and daily handling. However, these rats did not exhibit seizures. Rat Brain Tissue Preparation The animals in the epilepsy group were anesthetized using 3.5 % chloral hydrate (1 ml/100 g). A subset of the rats was decapitated, and the hippocampus and cortex were isolated and stored in liquid nitrogen for Western blotting. The remaining rats were perfused with saline and 4 % paraformaldehyde. The brains were removed and fixed in paraformaldehyde for 24 h. A portion of tissue from each fixed brain was frozen and sectioned at a thickness of 10 lm for immunofluorescence, and the remaining portion was fixed in paraffin and sectioned in paraffin at a thickness of 5 lm for immunohistochemistry. Experimental Methods Immunofluorescence Naturally dried and frozen sections were soaked in acetone for 30 min. Then, the sections were washed 3 times in PBS (5 min per wash) and wiped dry. They were then placed in a water bath containing 0.4 % Trion X-100 at 37 °C for 20 min, followed by incubation in goat serum in a 37 °C thermostatic water bath for 1 h to eliminate nonspecific staining. The serum was discarded, and the sections were incubated in a mixture of polyclonal rabbit anti-intersectin1-L (1:50, Abcam, catalog number ab118262) and monoclonal mouse anti-microtubule-associated protein2 (MAP2) antibodies (1:100, Zhongshan Golden Bridge, Beijing, China) or a mixture of polyclonal rabbit anti-intersectin1-L and mouse anti-glial fibrillary acidic protein (GFAP) antibodies (1:100, Zhongshan Golden Bridge, Beijing, China) overnight at 4 °C. On the second day, the sections were washed 3 times with PBS, a fluorescent secondary antibody mixture (DyLight 488 goat anti-rabbit IgG and DyLight 594 goat anti-mouse IgG) (1:50) was added, and the samples were incubated in a 37 °C water
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bath for 2 h. Then, the sections were sealed with a 1:1 glycerol:PBS mixture. Finally, images were captured via confocal microscopy (Leica Microsystems Heidelberg GmbH, Wetzlar, Germany). During this process, we shielded the sections from light to prevent the fluorescent secondary antibody mixture from interfering with the image during acquisition. Immunohistochemistry After dewaxing in xylene, the paraffinized sections were placed in graded ethanol solutions (100, 95, 80, and 75 %) for 5 min each. Then, the sections were incubated in 3 % H2O2 in a 37 °C water bath for 20 min. Next, 10 mmol/l citrate buffer was added, and the sections were heated in a microwave for 20 min for antigen retrieval. After the addition of goat serum, the sections were incubated for 30 min in a 37 °C water bath. The serum was discarded, and the slices were incubated with a polyclonal rabbit anti-intersectin1-L antibody at 4 °C overnight. Next, they were incubated with a goat anti-rabbit antibody (Zhongshan Golden Bridge, Beijing, China) for 30 min at 37 °C. Finally, they were incubated with a streptavidin-peroxidase complex (DAB; Zhongshan Golden Bridge, Beijing, China) under the same conditions. After the addition of DAB and the appearance of brown particles under the microscope, the slices were washed with distilled water to stop the reaction. Then, the sections were counterstained with hematoxylin. Finally, images were captured after dehydrating and transparently sealing the sections. The images were captured using an automated biological microscope (Olympus, Osaka, Japan). For each section, ten different random fields were imaged. A brown cell membrane indicated that a cell was positive for intersectin1-L expression. An average optical density value for each image was automatically measured by the computer. Intersectin1-L expression levels were quantitatively analyzed using the Image-Pro Plus 5.0 software. Western Blot After the brain tissue was homogenized, total protein was extracted according to the manufacturer’s instructions of the protein extraction reagent (Keygen Biotech, Nanjing, China). The protein concentration was measured according to the manufacturer’s instructions provided in the BCA Protein Concentration Assay Kit (Beyotime, Haimen, China). After denaturation, the protein samples were stored at -20 °C. To perform the Western blotting, a gel composed of a 5 % acrylamide separating gel and a 10 % acrylamide concentrating gel was prepared. Fifty micrograms of each protein sample was added to the lanes, and electrophoresis was performed for 60 min at 80 V. Then, the proteins were transferred to a PVDF membrane for 120 min, and membrane
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transfer was confirmed via Ponceau staining. The membranes were blocked using 5 % skim milk for 60 min at 37 °C. A polyclonal rabbit anti-intersectin1-L (1:600) or anti-GAPDH antibody (1:2000, Beyotime, China) was added to the membrane, which was incubated at 4 °C overnight. The following day, after washing with TBST, a secondary antibody (1:4000) was added, and the membrane was incubated at 37 °C for 1 h. The membrane was again washed, and then a chemical luminescence agent was added to enable the detection of the protein bands via digital scanning (Bio-Rad Laboratories). The optical density value of each band was quantitatively analyzed using the Quantity One software.
positive cellular staining, as revealed by brownish-yellow particles, was detected in the brain tissue of the patients with intractable epilepsy. The positive cells of the control group were scattered in buffy granules, and the negative control group, which was not treated with the polyclonal rabbit antiintersectin1-L antibody, did not display any positive cells. The average optical density of intersectin1-L in the epilepsy group was significantly higher than that in the control group (P \ 0.05). Thus, the expression of intersectin1-L in the brain tissue of the epilepsy group was higher than that in the brain tissue of the control group (Fig. 1c). Western Blotting
Statistical Analysis All data are presented as the mean ± standard deviation (SD). The SPSS 20.0 software was used for statistical analyses. Comparisons between the two groups were performed using Student’s t test. The parameters assessed included average age, gender, average optical density (immunohistochemistry), and the ratio of the optical densities (Western blot).
Results Expression of Intersectin1-L in the Brain Tissue of Patients with Intractable Epilepsy
The expression level of intersectin1-L in the brain tissue of the epilepsy patients was detected via Western blotting. GAPDH, which has a molecular weight of 36 kDa, was used as an internal loading control. Intersectin1-L and GAPDH protein bands were observed at molecular weights of approximately 195 and 36 kDa, respectively. These results supported the immunohistochemistry results of the expression of intersectin1-L based on the ratio of the optical density of the intersectin1-L protein band to that of the GAPDH protein band. The expression levels of intersectin1-L in the brain tissue of the epilepsy and control groups were compared using the t test, which revealed that the expression of intersectin1-L in the brain tissue of the epileptic patient group was higher than that in the brain tissue of the control group (P \ 0.05) (Fig. 1d).
Clinical Characteristics of the Patients The epileptic patient group consisted of 14 males and 8 females with an average age of 23.36 ± 7.00 years and a history of epilepsy for 8.5 ± 4.36 years. The control group consisted of 6 males and 4 females with an average age of 22.10 ± 6.37 years. The age and gender distributions of the two groups did not significantly differ (P [ 0.05). Immunofluorescence The localization of intersectin1-L expression was detected via double immunofluorescence in the brain tissue of the refractory epileptic patients. The results revealed that intersectin1-L (green) was co-expressed (yellow) with MAP2 (red) in the labeled neurons (Fig. 1a). However, intersectin1-L was not co-expressed with GFAP (red)-labeled glial cells (Fig. 1b). Therefore, this protein was primarily expressed in neurons but not in glial cells. Immunohistochemistry We performed immunohistochemistry to determine the expression of intersectin1-L in brain tissue. The results showed that it was primarily expressed in neurons. In addition,
The Expression of Intersectin1-L in the PilocarpineInduced TLE Animal Model (Fig. 2—Cortex and Fig. 3—Hippocampus) Immunofluorescence The localization of intersectin1-L expression was detected via double immunofluorescence in the cortex and hippocampus of the epilepsy animal model. The results showed the labeling of neurons with intersectin1-L (green) and MAP2 (red) and the co-expression of intersectin1-L and MAP2 (yellow) (Figs. 2a, 3a). However, intersectin1-L was not co-expressed with GFAP (red)-labeled glial cells (Figs. 2b, 3b). The intersectin1-L expression detected in the hippocampus was consistent with that observed in the cortex. Therefore, intersectin1-L was primarily expressed in neurons but not in glial cells. Immunohistochemistry We performed immunohistochemistry to determine the expression of intersectin1-L in the cortex and hippocampus of an animal model of TLE. The results showed that intersectin1-L was primarily expressed in neurons of the cortex and hippocampus. Moreover, positive cellular staining, as revealed by
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Cell Mol Neurobiol Fig. 1 The expression of intersectin1-L in the brain tissue of patients with intractable epilepsy. a Immunofluorescence: intersectin1-L (green) and MAP2 (red) were coexpressed (merged) in neurons. White arrow intersectin1-L?/MAP2? cell. b Intersectin1-L (green) and GFAP (red) were not coexpressed (merged). White arrow intersectin1-L? cell; blue arrow GFAP? cell. c Immunohistochemistry: black arrow intersectin1-L? cell. The strong immunoreactive staining of intersectin1-L is shown in the epilepsy group compared with light staining in control group. Scale bar 50 lm. The average optical density in the epilepsy group was significantly higher than that in the control group (P \ 0.05). d Western blot: intersectin1-L expression in the brain tissue of the epileptic patient group was higher than that in the brain tissue of the control group (P \ 0.05) (Color figure online)
brownish-yellow particles, was found in the hippocampus and cortex of the epileptic rats. The positive cellular staining of the control group was scattered in buffy granules, and no positive staining was detected in the negative control group, for which sections were not treated with the polyclonal rabbit anti-intersectin1-L antibody. The average optical density of intersectin1L in the epilepsy group was significantly higher than that in the control group (P \ 0.05). Thus, the expression of intersectin1L in the brain tissue of the epilepsy group was higher than that in the brain tissue of the control group (Figs. 2c, 3c). Western Blotting The expression level of intersectin1-L in the cortex and hippocampus of the TLE animal model was detected via Western blotting. GAPDH, which has a molecular weight of 36 kDa,
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was used as an internal loading control. Intersectin1-L and GAPDH protein bands were identified at molecular weights of approximately 195 and 36 kDa, respectively. These results indicated that the protein expression level of intersectin1-L in the TLE animal model animals was significantly higher than that in the control animals (P \ 0.05) (Figs. 2d, 3d). The Expression Changes of Intersectin1-L in a Kindled Animal Model of TLE The protein expression level of intersectin1-L was detected via Western blotting of the cortex (Fig. 4a) and hippocampus (Fig. 4b) tissue of the kindled TLE animal model. The results revealed that the expression of intersectin1-L in the epilepsy group was significantly higher than that in the control group (P \ 0.05).
Cell Mol Neurobiol Fig. 2 The expression of intersectin1-L in the cortex of the pilocarpine-induced TLE animal model. a Immunofluorescence: intersectin1-L (green) and MAP2 (red) were coexpressed (merged) in neurons. White arrow intersectin1-L?/MAP2? cell. b Intersectin1-L (green) and GFAP (red) were not coexpressed (merged). White arrow intersectin1-L? cell; blue arrow GFAP? cell. c Immunohistochemistry: Black arrow intersectin1-L? cell. Strong immunoreactive staining of intersectin1-L is shown in the epilepsy group compared with light staining in the control group. Scale bar 50 lm. The average optical density in the epilepsy group was significantly higher than that in the control group (P \ 0.05). d Western blot: intersectin1-L expression in the brain tissue of the epileptic patient group was higher than that in the brain tissue of the control group (P \ 0.05) (Color figure online)
Discussion There are several animal models of epilepsy that have been developed for various purposes. Each model is associated with distinct advantages for specific scientific studies. The lithium-pilocarpine-induced rat model of epilepsy induces specific changes in the selectively vulnerable hippocampal formation, which displays similar characteristics to human TLE. In recent years, this model has been broadly accepted as the most applicable animal model of TLE. In 1969, Goddard et al. (1969) first established the electrical kindling model. Two of the most classic and popular stimulation sites are the hippocampus and amygdala (Barnes and Pinel 2001). The amygdalar pathology exhibited by the kindling model is similar to the pathological changes observed in human TLE. The kindling epilepsy model is currently widely used in studies of TLE (Morimoto et al. 2004). Two animal models were used for this study. Neurotransmitters are stored in vesicles in nerve
endings and are released into the synaptic cleft via exocytosis. After neurotransmitter release, the vesicle is taken back up by the nerve ending via endocytosis to prepare for the next neurotransmitter release event. Abnormalities in neurotransmitter release at any stage may cause abnormal neuronal discharges, which could ultimately lead to the development of epilepsy. As a scaffold protein, intersectin performs a variety of functions due to its many domains that interact with other proteins. One study has found that intersectin1-L plays an important role in synaptic vesicle recycling activity (Koh et al. 2004). Intersectin1-L is expressed in all brain regions and is most abundantly expressed in hippocampal and cortical neurons. Intersectin and endocytosis- and exocytosis-related proteins, such as dynamin, endophilin, and synaptojanin, are often co-expressed (Marie et al. 2004; Okamoto et al. 1999). Intersectin is an essential component of synaptic vesicle recycling (Ma et al. 2003; Yu et al. 2008) and is involved in the processes of endocytosis and
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Cell Mol Neurobiol Fig. 3 The expression of intersectin1-L in the hippocampus of the pilocarpineinduced TLE animal model. a Immunofluorescence: intersectin1-L (green) and MAP2 (red) were coexpressed (merged) in neurons. White arrow intersectin1-L?/MAP2? cell. b intersectin1-L (green) and GFAP (red) were not coexpressed (merged). White arrow intersectin1-L? cell; blue arrow GFAP? cell. c Immunohistochemistry: black arrow intersectin1-L? cell. Strong immunoreactive staining of intersectin1-L is shown in the epilepsy group compared with light staining in the control group. Scale bar 50 lm. The average optical density in the epilepsy group was significantly higher than that in the control group (P \ 0.05). d Western blot: intersectin1-L expression in the brain tissue of the epileptic patient group was higher than that in brain tissue of the control group (P \ 0.05) (Color figure online)
Fig. 4 The expression changes of intersectin1-L in a kindled animal model of TLE. Western blot: the results revealed that the expression levels of intersectin1-L in the cortex (a) and hippocampus (b) of the epilepsy group were significantly higher those in the control group (P \ 0.05)
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exocytosis in neurons (Evergren et al. 2007; Gubar et al. 2013; Morderer et al. 2012). Furthermore, one study has reported that intersectin1-L is involved in the regulation of rapid neurotransmitter release (Sakaba et al. 2013). An additional study has associated intersectin1-L with N-type Ca2? channels (Khanna et al. 2007). Interestingly, the upregulation of N-type Ca2? channels has been reported in kindling epileptogenesis (Bernstein et al. 1999). In the present study, the expression of intersectin1-L in intractable epilepsy was evaluated. The immunofluorescence and immunohistochemistry results indicated that this protein is primarily expressed in neurons, which is consistent with previous reports. The quantification of intersectin1-L expression via immunohistochemical and Western blot analyses revealed that the brain tissue from the patients with epilepsy displayed significantly higher expression than that in the brain tissue from the controls. To eliminate effects of drugs or other possible factors that may have led to these experimental results, they were verified using an animal model of epilepsy induced by the drug pilocarpine. The animal model results showed that intersectin1-L was primarily expressed in neurons and that its expression was significantly higher in the epilepsy group than in the control group. These results were confirmed by detecting intersectin1-L levels in the brain tissue of kindled epilepsy model animals and were consistent with the expression changes observed in the brain tissue of patients with intractable epilepsy and the pilocarpine-induced epilepsy model animals, indicating that this protein may affect the release of neurotransmitters involved in epileptogenesis. Further studies will be performed to explore the role of intersectin1-L in the development of epilepsy. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81271445 and 81301110). We would like to acknowledge Xuanwu Hospital, Capital Medical University, Tiantan Hospital, the Third Military Medical University, Xinqiao Hospital and the First Affiliated Hospital of Chongqing Medical University, which provided the brain tissue samples. We also thank the patients and their families who participated in this study. Conflict of interest
All authors report no conflicts of interest.
Ethical standard This study was approved by the Ethics Committee of Chongqing Medical University. The entire study protocol was in compliance with the Declaration of Helsinki of the World Medical Association guidelines. All efforts were made to minimize the number of animals used and their suffering.
References Barnes SJ, Pinel JP (2001) Conditioned effects of kindling. Neurosci Biobehav Rev 25:745–751 Beleza P (2009) Refractory epilepsy: a clinically oriented review European neurology 62:65–71. doi:10.1159/000222775
Bernstein GM, Mendonca A, Wadia J, Burnham WM, Jones OT (1999) Kindling induces a long-term enhancement in the density of N-type calcium channels in the rat hippocampus. Neuroscience 94:1083–1095 Dulac O, Milh M (2012) Brain maturation and epilepsy. La Revue du praticien 62:1371–1377 Evergren E, Gad H, Walther K, Sundborger A, Tomilin N, Shupliakov O (2007) Intersectin is a negative regulator of dynamin recruitment to the synaptic endocytic zone in the central synapse. J Neurosci 27:379–390. doi:10.1523/jneurosci. 4683-06.2007 Goddard GV, McIntyre DC, Leech CK (1969) A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol 25:295–330 Gubar O et al (2013) Intersectin: the crossroad between vesicle exocytosis and endocytosis. Front Endocrinol 4:109. doi:10. 3389/fendo.2013.00109 Khanna R, Li Q, Schlichter LC, Stanley EF (2007) The transmitter release-site CaV2.2 channel cluster is linked to an endocytosis coat protein complex. Eur J Neurosci 26:560–574. doi:10.1111/j. 1460-9568.2007.05681.x Koh TW, Verstreken P, Bellen HJ (2004) Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis. Neuron 43:193–205. doi:10.1016/j.neuron. 2004.06.029 Kropyvko S et al (2010) Structural diversity and differential expression of novel human intersectin 1 isoforms. Mol Biol Rep 37:2789–2796. doi:10.1007/s11033-009-9824-8 Kwan P, Brodie MJ (2000) Early identification of refractory epilepsy. New Engl J Med 342:314–319. doi:10.1056/ NEJM200002033420503 Leach JP, Abassi H (2013) Mod Manage Epilepsy Clin Med 13:84–86 Ma YJ et al (2003) Neuronal distribution of EHSH1/intersectin: molecular linker between clathrin-mediated endocytosis and signaling pathways. J Neurosci Res 71:468–477. doi:10.1002/jnr. 10500 Ma Y, Wang B, Li W, Ying G, Fu L, Niu R, Gu F (2010) Reduction of intersectin1-s induced apoptosis of human glioblastoma cells. Brain Res 1351:222–228. doi:10.1016/j.brainres.2010.05.028 Ma Y et al (2011) Intersectin1-s is involved in migration and invasion of human glioma cells. J Neurosci Res 89:1079–1090. doi:10. 1002/jnr.22616 Madsen TM, Bolwig TG, Mikkelsen JD (2006) Differential regulation of c-Fos and FosB in the rat brain after amygdala kindling. Cell Mol Neurobiol 26:87–100. doi:10.1007/s10571-006-9202-1 Marie B, Sweeney ST, Poskanzer KE, Roos J, Kelly RB, Davis GW (2004) Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth. Neuron 43:207–219. doi:10.1016/j.neuron.2004.07.001 Morderer D, Nikolaienko O, Skrypkina I, Cherkas V, Tsyba L, Belan P, Rynditch A (2012) Endocytic adaptor protein intersectin 1 forms a complex with microtubule stabilizer STOP in neurons. Gene 505:360–364. doi:10.1016/j.gene.2012.06.061 Morimoto K, Fahnestock M, Racine RJ (2004) Kindling and status epilepticus models of epilepsy: rewiring the brain. Prog Neurobiol 73:1–60. doi:10.1016/j.pneurobio.2004.03.009 O’Bryan JP, Mohney RP, Oldham CE (2001) Mitogenesis and endocytosis: What’s at the INTERSECTIoN? Oncogene 20:6300–6308. doi:10.1038/sj.onc.1204773 Okamoto M, Schoch S, Sudhof TC (1999) EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis? J Biol Chem 274:18446–18454 Pitka¨nen A, Schwartzkroin PA, Moshe´ SL (2005) Models of seizures and epilepsy. Academic Press, New York
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ORIGINAL RESEARCH
Altered Expression of Intersectin1-L in Patients with Refractory Epilepsy and in Experimental Epileptic Rats Xiaoyan Yang1 • Xin Xu1 • Yujiao Zhang1 • Shasha Wang2 • Minghui Li2 Xuefeng Wang1
•
Received: 15 December 2014 / Accepted: 11 March 2015 Ó Springer Science+Business Media New York 2015
Abstract Epilepsy is a common neurological disorder. Because its underlying mechanisms remain incompletely understood, current treatments are not adequate for all epilepsy patients, and some patients progress to refractory epilepsy. Under physiological conditions, excitatory and inhibitory neurons function in a dynamic balance. Epilepsy develops when this balance is disrupted. Intersectin1-L is a major scaffold protein in the central nervous system that contains multiple functional domains, and it is the long form of intersectin1. Recent studies have shown that intersectin1-L plays an important role in the process of neurotransmitter release. In this study, we investigated the expression pattern and distribution of intersectin1-L in patients with refractory epilepsy, in a rat model of pilocarpine-induced epilepsy, and in a rat model of amygdalakindled epilepsy by immunohistochemistry, immunofluorescence, and Western blotting. The purpose of this study was to explore the relationship between epilepsy and intersectin1-L. The results showed that the intersectin1-L protein was primarily expressed in neurons in brain tissue. Its expression was remarkably increased in patients with refractory epilepsy and in epilepsy model rats. These results suggest that the abnormal expression of the intersectin1-L protein in epileptic brain tissue may play an important role in epilepsy, especially refractory epilepsy. Xiaoyan Yang and Xin Xu have contributed equally to this work. & Xuefeng Wang [email protected] 1
Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Chongqing 400016, China
2
Chongqing Medical University, No.1 Yixueyuan Road, Chongqing 400016, China
Keywords
Intersectin1-L Patient Epilepsy Rat model
Introduction Epilepsy is a common disease of the nervous system with a high incidence. Approximately 50 million people suffer from epilepsy worldwide (Dulac and Milh 2012), and onethird of epilepsy patients develop refractory epilepsy after unsuccessful drug treatment. Repeated seizures may cause neuronal damage, resulting in irreversible brain damage and poor prognosis (Towne 2007). Epilepsy causes substantial economic and psychological burdens on patients and their families. The pathology of epilepsy is based on the abnormal synchronization of neuronal discharges (Leach and Abassi 2013). However, the mechanism underlying refractory epilepsy remains to be fully elucidated. Under physiological conditions, neuronal excitation and inhibition are maintained in dynamic equilibrium. This balance can be disrupted by abnormal neurotransmitter release. Altered neuronal excitability causes abnormal discharges, which may lead to the development of epilepsy. Intersectin is a scaffold protein that contains multiple functional domains (O’Bryan et al. 2001). The human intersectin gene encodes two intersectin proteins, intersectin1 and intersectin2. Each protein has two isoforms, referred to as intersectin-L and intersectin-S (Kropyvko et al. 2010; Yu et al. 2008), which are expressed in tissuespecific manners. Intersectin1-L is primarily expressed in neurons of the central nervous system (Ma et al. 2003) and plays an important role in synaptic neurotransmitter release (Ma et al. 2003; Sakaba et al. 2013). Intersectin1-S is primarily expressed in glial cells and plays important roles in cell apoptosis and cytoskeleton reorganization (Ma et al. 2010, 2011).
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Temporal lobe epilepsy (TLE) is the most common type of refractory epilepsy (Beleza 2009; Kwan and Brodie 2000). Considering the localization of intersectin1-L expression and its regulatory function in neurotransmitter release, we hypothesized that its expression in the brain tissue of refractory epilepsy patients and TLE animal models may be altered and that such alterations may be involved in the development of refractory epilepsy. To test this hypothesis, we performed immunofluorescence, immunohistochemistry, and Western blot analyses to detect the expression of intersectin1-L in the brain tissues of patients with refractory epilepsy and of animal models of TLE.
Materials and Methods All patients enrolled in this study provided written informed consent. Human brain tissue samples were obtained from Xuanwu Hospital, Capital Medical University, Tiantan Hospital, the Third Military Medical University, Xinqiao Hospital and the First Affiliated Hospital of Chongqing Medical University. This study was approved by the Ethics Committee of Chongqing Medical University. The entire study protocol was in compliance with the Declaration of Helsinki of the World Medical Association guidelines. Human Brain Tissue Human Brain Tissue and Clinical Data We constructed a brain tissue library comprising 223 brain tissue samples from patients with refractory TLE and 60 brain tissue samples from a control group of patients. The epilepsy patients exhibited typical clinical characteristics and changes in their EEG, and they met the 2001 International League Against Epilepsy diagnostic criteria for epilepsy. In these patients, treatment with the maximum doses of three types of antiepileptic drugs was unsuccessful, and surgery was elected. Preoperative evaluation included a detailed medical history, neurological examination, radiographic examination, and routine preoperative examination. The epileptic focus was localized via video EEG or 24-h ambulatory EEG prior to surgery, and surgery was performed during sphenoidal electrode monitoring and intraoperative electrocorticography. After excision of the epileptic focus, EEG of the perilesional tissue was performed to verify the complete excision of the lesion. The pathological assessment of the excised lesion revealed neuronal degeneration or gliosis, which is consistent with the pathological manifestations of epilepsy. The brain tissue of the control group was obtained from a
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resected area that was excised during decompression after a traumatic brain injury. The results of the pathological assessment of the brain tissue from the control group were normal. The clinical records of the control patients indicated that they had no history of epilepsy or the use of antiepileptic medication. For the present study, we randomly selected 22 samples from the epilepsy patient group and 10 samples from the control group from the brain tissue library. The clinical data are shown in Tables 1 and 2. Human Brain Tissue Preparation The collected specimens were divided into three groups. One group of specimens was stored in liquid nitrogen for Western blotting to detect the protein expression of intersectin1-L. The remaining two groups of specimens were immediately fixed with 4 % phosphate-buffered formalin for 48 h. After fixation, one of these groups of specimens was frozen and sectioned at a thickness of 10 lm for immunofluorescence to determine the localization of intersectin1-L expression. The remaining group was sectioned in paraffin at a thickness of 5 lm for immunohistochemistry to detect the expression of intersectin1-L. Brain tissue from Animal Models of Epilepsy Animal Selection and Breeding Healthy adult Sprague–Dawley rats (7–8 weeks old, 200–240 g) provided by the Chongqing Medical University Animal Center were used for this experiment. The animals were housed at 24–26 °C with a relative humidity of 50–60 % under an artificial 12 h light–dark cycle to maintain their circadian rhythm and were provided with free access to food and water. Pilocarpine-Induced Animal Model of Epilepsy Sixteen healthy adult rats were randomly assigned to a control group or epilepsy group (n = 8). Each rat in the epilepsy group was administered 127 mg/kg lithium chloride (Sigma, USA) and after 12 h, received 50 mg/kg pilocarpine (Sigma, USA) via intraperitoneal injection. Because status epilepticus (SE) may not always appear after the first injection of pilocarpine, the treatment was repeatedly administered until the onset of SE. In most cases, pilocarpine administration was repeated 5 times, with 30 min intervals between each injection. After the onset of SE, seizures were rated from 1 to 5 according to Racine’s scale. Animals receiving a score of 4 or higher were included in follow-up studies. For cases in which SE exceeded 60 min, diazepam (10 mg/kg) (Sigma, USA) was administered to terminate the seizures. After the
Cell Mol Neurobiol Table 1 Clinical characteristics of intractable epilepsy patients Patient
Sex (M/F)
Age (years)
Course (years)
AEDs before surgery
Source of organization
Pathologic result
1
M
15
7
LTG/TPM/CBZ
RTN
NL/G
2
F
32
16
VPA/PB/CBZ
LTN
NL/G
3
M
18
4
CBZ/VPA/PHT
RTN
NL/G
4
F
22
6
VPA/CBZ/PB
RTN
NL/G
5
M
16
3
VPA/TPM/CBZ
RTN
G
6
F
35
18
VPA/PB/CBZ/LEV
LTN
NL/G
7
M
17
5
VPA/CBZ//TPM
LTN
NL/G
8
M
23
5
CBZ/VPA/PHT/PB
LTN
NL/G
9
M
26
12
PHT/VPA/PB/TPM
LTN
NL/G
10
F
16
6
CBZ/LEV/TPM/LTG
RTN
NL/G
11 12
M M
25 16
8 4
VPACBZ/TPM VPA/OXC/TPM/PB
RTN LTN
NL/G G
13
M
18
3
VPA/CBZ/TP
RTN
G
14
F
21
7
CBZ/PB/LTG/LEV
RTN
NL/G
15
F
28
11
CBZ/PHT/PB/LTG
LTN
NL/G
16
M
24
12
CBZ/PHT/VPA/PB
LTN
NL/G
17
M
19
8
VPA/CBZ/PB
RTN
NL/G
18
F
20
5
CBZ/LTG/LEV
RTN
NL/G
19
M
39
15
CBZ/VPA/PHT/PB
RTN
NL/G
20
M
36
12
VPA/PHT/CBZ/PB
LTN
NL/G
21
M
23
8
VPA/CBZ/PHT
LTN
NL/G
22
F
25
12
CBZ/PB/LTG/LEV
LTN
NL/G
F female, M male, AEDs antiepilepti drugs, LTG lamotrigine, TPM topiramate, CBZ carbamazepine, PB phenobarbital, VPA valproic acid, LEV levetiracetam, OXC oxcarbazepine, RTN right temporal neocortex, LTN left temporal neocortex, NL neuronal necrosis, G gliosis
Table 2 Clinical characteristics of control group
Patient
Sex
Age (years)
Disease diagnosis
Source of organization
Pathologic result
1
M
25
Brain trauma
LTN
Normal
2
M
19
Brain trauma
LTN
Normal
3
M
36
Brain trauma
RTN
Normal
4
M
14
Brain trauma
RTN
Normal
5 6
F F
23 21
Brain trauma Brain trauma
LTN RTN
Normal Normal
7
M
16
Brain trauma
LTN
Normal
8
F
28
Brain trauma
RTN
Normal
9
M
19
Brain trauma
RTN
Normal
10
F
20
Brain trauma
LTN
Normal
F female, M male, RTN right temporal neocortex, LTN light temporal neocortex
termination of SE, we observed whether spontaneous seizures occurred in the rats during the chronic phase. Rats exhibiting spontaneous seizures were included in the epilepsy group. The control group of rats was injected with normal saline instead of lithium chloride and pilocarpine and underwent an identical surgical procedure.
Amygdala-Kindled Epilepsy Animal Model Sixteen healthy adult rats were randomly assigned to a control group or epilepsy group (n = 8), and 3.5 % chloral hydrate was administered via intraperitoneal injection at 1 ml/100 g by weight. The rats were anesthetized and fixed to a stereotaxic frame. The skull was fully exposed, and the
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position of the amygdala was located (2 mm posterior and 5 mm lateral to the bregma at a depth of 8 mm). After drilling through the skull, an electrode was implanted and fixed using dental cement. The postoperative rats were reared under the same conditions as the preoperative rats. Stimulation was initiated at 8 days after the surgical procedure (stimulation parameters: train stimulation, square wave, and wave width of -1 ms, and frequency of -50 Hz). The rats were stimulated once daily for 10 consecutive days. The model was considered to be successful when the rats exhibited seizures (according to Racine’s scale, Cstage 4) (Madsen et al. 2006; Pitka¨nen et al. 2005). The control group underwent stereotactic surgery and daily handling. However, these rats did not exhibit seizures. Rat Brain Tissue Preparation The animals in the epilepsy group were anesthetized using 3.5 % chloral hydrate (1 ml/100 g). A subset of the rats was decapitated, and the hippocampus and cortex were isolated and stored in liquid nitrogen for Western blotting. The remaining rats were perfused with saline and 4 % paraformaldehyde. The brains were removed and fixed in paraformaldehyde for 24 h. A portion of tissue from each fixed brain was frozen and sectioned at a thickness of 10 lm for immunofluorescence, and the remaining portion was fixed in paraffin and sectioned in paraffin at a thickness of 5 lm for immunohistochemistry. Experimental Methods Immunofluorescence Naturally dried and frozen sections were soaked in acetone for 30 min. Then, the sections were washed 3 times in PBS (5 min per wash) and wiped dry. They were then placed in a water bath containing 0.4 % Trion X-100 at 37 °C for 20 min, followed by incubation in goat serum in a 37 °C thermostatic water bath for 1 h to eliminate nonspecific staining. The serum was discarded, and the sections were incubated in a mixture of polyclonal rabbit anti-intersectin1-L (1:50, Abcam, catalog number ab118262) and monoclonal mouse anti-microtubule-associated protein2 (MAP2) antibodies (1:100, Zhongshan Golden Bridge, Beijing, China) or a mixture of polyclonal rabbit anti-intersectin1-L and mouse anti-glial fibrillary acidic protein (GFAP) antibodies (1:100, Zhongshan Golden Bridge, Beijing, China) overnight at 4 °C. On the second day, the sections were washed 3 times with PBS, a fluorescent secondary antibody mixture (DyLight 488 goat anti-rabbit IgG and DyLight 594 goat anti-mouse IgG) (1:50) was added, and the samples were incubated in a 37 °C water
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bath for 2 h. Then, the sections were sealed with a 1:1 glycerol:PBS mixture. Finally, images were captured via confocal microscopy (Leica Microsystems Heidelberg GmbH, Wetzlar, Germany). During this process, we shielded the sections from light to prevent the fluorescent secondary antibody mixture from interfering with the image during acquisition. Immunohistochemistry After dewaxing in xylene, the paraffinized sections were placed in graded ethanol solutions (100, 95, 80, and 75 %) for 5 min each. Then, the sections were incubated in 3 % H2O2 in a 37 °C water bath for 20 min. Next, 10 mmol/l citrate buffer was added, and the sections were heated in a microwave for 20 min for antigen retrieval. After the addition of goat serum, the sections were incubated for 30 min in a 37 °C water bath. The serum was discarded, and the slices were incubated with a polyclonal rabbit anti-intersectin1-L antibody at 4 °C overnight. Next, they were incubated with a goat anti-rabbit antibody (Zhongshan Golden Bridge, Beijing, China) for 30 min at 37 °C. Finally, they were incubated with a streptavidin-peroxidase complex (DAB; Zhongshan Golden Bridge, Beijing, China) under the same conditions. After the addition of DAB and the appearance of brown particles under the microscope, the slices were washed with distilled water to stop the reaction. Then, the sections were counterstained with hematoxylin. Finally, images were captured after dehydrating and transparently sealing the sections. The images were captured using an automated biological microscope (Olympus, Osaka, Japan). For each section, ten different random fields were imaged. A brown cell membrane indicated that a cell was positive for intersectin1-L expression. An average optical density value for each image was automatically measured by the computer. Intersectin1-L expression levels were quantitatively analyzed using the Image-Pro Plus 5.0 software. Western Blot After the brain tissue was homogenized, total protein was extracted according to the manufacturer’s instructions of the protein extraction reagent (Keygen Biotech, Nanjing, China). The protein concentration was measured according to the manufacturer’s instructions provided in the BCA Protein Concentration Assay Kit (Beyotime, Haimen, China). After denaturation, the protein samples were stored at -20 °C. To perform the Western blotting, a gel composed of a 5 % acrylamide separating gel and a 10 % acrylamide concentrating gel was prepared. Fifty micrograms of each protein sample was added to the lanes, and electrophoresis was performed for 60 min at 80 V. Then, the proteins were transferred to a PVDF membrane for 120 min, and membrane
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transfer was confirmed via Ponceau staining. The membranes were blocked using 5 % skim milk for 60 min at 37 °C. A polyclonal rabbit anti-intersectin1-L (1:600) or anti-GAPDH antibody (1:2000, Beyotime, China) was added to the membrane, which was incubated at 4 °C overnight. The following day, after washing with TBST, a secondary antibody (1:4000) was added, and the membrane was incubated at 37 °C for 1 h. The membrane was again washed, and then a chemical luminescence agent was added to enable the detection of the protein bands via digital scanning (Bio-Rad Laboratories). The optical density value of each band was quantitatively analyzed using the Quantity One software.
positive cellular staining, as revealed by brownish-yellow particles, was detected in the brain tissue of the patients with intractable epilepsy. The positive cells of the control group were scattered in buffy granules, and the negative control group, which was not treated with the polyclonal rabbit antiintersectin1-L antibody, did not display any positive cells. The average optical density of intersectin1-L in the epilepsy group was significantly higher than that in the control group (P \ 0.05). Thus, the expression of intersectin1-L in the brain tissue of the epilepsy group was higher than that in the brain tissue of the control group (Fig. 1c). Western Blotting
Statistical Analysis All data are presented as the mean ± standard deviation (SD). The SPSS 20.0 software was used for statistical analyses. Comparisons between the two groups were performed using Student’s t test. The parameters assessed included average age, gender, average optical density (immunohistochemistry), and the ratio of the optical densities (Western blot).
Results Expression of Intersectin1-L in the Brain Tissue of Patients with Intractable Epilepsy
The expression level of intersectin1-L in the brain tissue of the epilepsy patients was detected via Western blotting. GAPDH, which has a molecular weight of 36 kDa, was used as an internal loading control. Intersectin1-L and GAPDH protein bands were observed at molecular weights of approximately 195 and 36 kDa, respectively. These results supported the immunohistochemistry results of the expression of intersectin1-L based on the ratio of the optical density of the intersectin1-L protein band to that of the GAPDH protein band. The expression levels of intersectin1-L in the brain tissue of the epilepsy and control groups were compared using the t test, which revealed that the expression of intersectin1-L in the brain tissue of the epileptic patient group was higher than that in the brain tissue of the control group (P \ 0.05) (Fig. 1d).
Clinical Characteristics of the Patients The epileptic patient group consisted of 14 males and 8 females with an average age of 23.36 ± 7.00 years and a history of epilepsy for 8.5 ± 4.36 years. The control group consisted of 6 males and 4 females with an average age of 22.10 ± 6.37 years. The age and gender distributions of the two groups did not significantly differ (P [ 0.05). Immunofluorescence The localization of intersectin1-L expression was detected via double immunofluorescence in the brain tissue of the refractory epileptic patients. The results revealed that intersectin1-L (green) was co-expressed (yellow) with MAP2 (red) in the labeled neurons (Fig. 1a). However, intersectin1-L was not co-expressed with GFAP (red)-labeled glial cells (Fig. 1b). Therefore, this protein was primarily expressed in neurons but not in glial cells. Immunohistochemistry We performed immunohistochemistry to determine the expression of intersectin1-L in brain tissue. The results showed that it was primarily expressed in neurons. In addition,
The Expression of Intersectin1-L in the PilocarpineInduced TLE Animal Model (Fig. 2—Cortex and Fig. 3—Hippocampus) Immunofluorescence The localization of intersectin1-L expression was detected via double immunofluorescence in the cortex and hippocampus of the epilepsy animal model. The results showed the labeling of neurons with intersectin1-L (green) and MAP2 (red) and the co-expression of intersectin1-L and MAP2 (yellow) (Figs. 2a, 3a). However, intersectin1-L was not co-expressed with GFAP (red)-labeled glial cells (Figs. 2b, 3b). The intersectin1-L expression detected in the hippocampus was consistent with that observed in the cortex. Therefore, intersectin1-L was primarily expressed in neurons but not in glial cells. Immunohistochemistry We performed immunohistochemistry to determine the expression of intersectin1-L in the cortex and hippocampus of an animal model of TLE. The results showed that intersectin1-L was primarily expressed in neurons of the cortex and hippocampus. Moreover, positive cellular staining, as revealed by
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Cell Mol Neurobiol Fig. 1 The expression of intersectin1-L in the brain tissue of patients with intractable epilepsy. a Immunofluorescence: intersectin1-L (green) and MAP2 (red) were coexpressed (merged) in neurons. White arrow intersectin1-L?/MAP2? cell. b Intersectin1-L (green) and GFAP (red) were not coexpressed (merged). White arrow intersectin1-L? cell; blue arrow GFAP? cell. c Immunohistochemistry: black arrow intersectin1-L? cell. The strong immunoreactive staining of intersectin1-L is shown in the epilepsy group compared with light staining in control group. Scale bar 50 lm. The average optical density in the epilepsy group was significantly higher than that in the control group (P \ 0.05). d Western blot: intersectin1-L expression in the brain tissue of the epileptic patient group was higher than that in the brain tissue of the control group (P \ 0.05) (Color figure online)
brownish-yellow particles, was found in the hippocampus and cortex of the epileptic rats. The positive cellular staining of the control group was scattered in buffy granules, and no positive staining was detected in the negative control group, for which sections were not treated with the polyclonal rabbit anti-intersectin1-L antibody. The average optical density of intersectin1L in the epilepsy group was significantly higher than that in the control group (P \ 0.05). Thus, the expression of intersectin1L in the brain tissue of the epilepsy group was higher than that in the brain tissue of the control group (Figs. 2c, 3c). Western Blotting The expression level of intersectin1-L in the cortex and hippocampus of the TLE animal model was detected via Western blotting. GAPDH, which has a molecular weight of 36 kDa,
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was used as an internal loading control. Intersectin1-L and GAPDH protein bands were identified at molecular weights of approximately 195 and 36 kDa, respectively. These results indicated that the protein expression level of intersectin1-L in the TLE animal model animals was significantly higher than that in the control animals (P \ 0.05) (Figs. 2d, 3d). The Expression Changes of Intersectin1-L in a Kindled Animal Model of TLE The protein expression level of intersectin1-L was detected via Western blotting of the cortex (Fig. 4a) and hippocampus (Fig. 4b) tissue of the kindled TLE animal model. The results revealed that the expression of intersectin1-L in the epilepsy group was significantly higher than that in the control group (P \ 0.05).
Cell Mol Neurobiol Fig. 2 The expression of intersectin1-L in the cortex of the pilocarpine-induced TLE animal model. a Immunofluorescence: intersectin1-L (green) and MAP2 (red) were coexpressed (merged) in neurons. White arrow intersectin1-L?/MAP2? cell. b Intersectin1-L (green) and GFAP (red) were not coexpressed (merged). White arrow intersectin1-L? cell; blue arrow GFAP? cell. c Immunohistochemistry: Black arrow intersectin1-L? cell. Strong immunoreactive staining of intersectin1-L is shown in the epilepsy group compared with light staining in the control group. Scale bar 50 lm. The average optical density in the epilepsy group was significantly higher than that in the control group (P \ 0.05). d Western blot: intersectin1-L expression in the brain tissue of the epileptic patient group was higher than that in the brain tissue of the control group (P \ 0.05) (Color figure online)
Discussion There are several animal models of epilepsy that have been developed for various purposes. Each model is associated with distinct advantages for specific scientific studies. The lithium-pilocarpine-induced rat model of epilepsy induces specific changes in the selectively vulnerable hippocampal formation, which displays similar characteristics to human TLE. In recent years, this model has been broadly accepted as the most applicable animal model of TLE. In 1969, Goddard et al. (1969) first established the electrical kindling model. Two of the most classic and popular stimulation sites are the hippocampus and amygdala (Barnes and Pinel 2001). The amygdalar pathology exhibited by the kindling model is similar to the pathological changes observed in human TLE. The kindling epilepsy model is currently widely used in studies of TLE (Morimoto et al. 2004). Two animal models were used for this study. Neurotransmitters are stored in vesicles in nerve
endings and are released into the synaptic cleft via exocytosis. After neurotransmitter release, the vesicle is taken back up by the nerve ending via endocytosis to prepare for the next neurotransmitter release event. Abnormalities in neurotransmitter release at any stage may cause abnormal neuronal discharges, which could ultimately lead to the development of epilepsy. As a scaffold protein, intersectin performs a variety of functions due to its many domains that interact with other proteins. One study has found that intersectin1-L plays an important role in synaptic vesicle recycling activity (Koh et al. 2004). Intersectin1-L is expressed in all brain regions and is most abundantly expressed in hippocampal and cortical neurons. Intersectin and endocytosis- and exocytosis-related proteins, such as dynamin, endophilin, and synaptojanin, are often co-expressed (Marie et al. 2004; Okamoto et al. 1999). Intersectin is an essential component of synaptic vesicle recycling (Ma et al. 2003; Yu et al. 2008) and is involved in the processes of endocytosis and
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Cell Mol Neurobiol Fig. 3 The expression of intersectin1-L in the hippocampus of the pilocarpineinduced TLE animal model. a Immunofluorescence: intersectin1-L (green) and MAP2 (red) were coexpressed (merged) in neurons. White arrow intersectin1-L?/MAP2? cell. b intersectin1-L (green) and GFAP (red) were not coexpressed (merged). White arrow intersectin1-L? cell; blue arrow GFAP? cell. c Immunohistochemistry: black arrow intersectin1-L? cell. Strong immunoreactive staining of intersectin1-L is shown in the epilepsy group compared with light staining in the control group. Scale bar 50 lm. The average optical density in the epilepsy group was significantly higher than that in the control group (P \ 0.05). d Western blot: intersectin1-L expression in the brain tissue of the epileptic patient group was higher than that in brain tissue of the control group (P \ 0.05) (Color figure online)
Fig. 4 The expression changes of intersectin1-L in a kindled animal model of TLE. Western blot: the results revealed that the expression levels of intersectin1-L in the cortex (a) and hippocampus (b) of the epilepsy group were significantly higher those in the control group (P \ 0.05)
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exocytosis in neurons (Evergren et al. 2007; Gubar et al. 2013; Morderer et al. 2012). Furthermore, one study has reported that intersectin1-L is involved in the regulation of rapid neurotransmitter release (Sakaba et al. 2013). An additional study has associated intersectin1-L with N-type Ca2? channels (Khanna et al. 2007). Interestingly, the upregulation of N-type Ca2? channels has been reported in kindling epileptogenesis (Bernstein et al. 1999). In the present study, the expression of intersectin1-L in intractable epilepsy was evaluated. The immunofluorescence and immunohistochemistry results indicated that this protein is primarily expressed in neurons, which is consistent with previous reports. The quantification of intersectin1-L expression via immunohistochemical and Western blot analyses revealed that the brain tissue from the patients with epilepsy displayed significantly higher expression than that in the brain tissue from the controls. To eliminate effects of drugs or other possible factors that may have led to these experimental results, they were verified using an animal model of epilepsy induced by the drug pilocarpine. The animal model results showed that intersectin1-L was primarily expressed in neurons and that its expression was significantly higher in the epilepsy group than in the control group. These results were confirmed by detecting intersectin1-L levels in the brain tissue of kindled epilepsy model animals and were consistent with the expression changes observed in the brain tissue of patients with intractable epilepsy and the pilocarpine-induced epilepsy model animals, indicating that this protein may affect the release of neurotransmitters involved in epileptogenesis. Further studies will be performed to explore the role of intersectin1-L in the development of epilepsy. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 81271445 and 81301110). We would like to acknowledge Xuanwu Hospital, Capital Medical University, Tiantan Hospital, the Third Military Medical University, Xinqiao Hospital and the First Affiliated Hospital of Chongqing Medical University, which provided the brain tissue samples. We also thank the patients and their families who participated in this study. Conflict of interest
All authors report no conflicts of interest.
Ethical standard This study was approved by the Ethics Committee of Chongqing Medical University. The entire study protocol was in compliance with the Declaration of Helsinki of the World Medical Association guidelines. All efforts were made to minimize the number of animals used and their suffering.
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