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Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis
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Research Report
Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis Q1
Yuanyuan Luoa, Qiao Hua, Qian Zhanga, Siqi Honga,b, Xiaoju Tanga, Li Chena, Li Jianga,b,n a Lab of Pediatric Neurology, Ministry of Education, Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical University, Chongqing, PR China b Department of Neurology, Children’s Hospital of Chongqing Medical University, 136# Zhongshan 2 Road, Chongqing 400014, PR China
art i cle i nfo
ab st rac t
Article history:
Recent reports have described damage to myelinated fibers in the central nervous system
Accepted 22 September 2015
(CNS) in patients with temporal lobe epilepsy (TLE) and animal models. However, only limited data are available on the dynamic changes that occur in myelinated fibers, oligodendrocytes (which are myelin-forming cells), and oligodendrocyte precursor cells
Keywords:
(OPCs), which are a reservoir of new oligodendrocytes, in the hippocampus throughout
Myelin sheath Oligodendrocyte precursor cells Oligodendrocyte
epileptogenesis. The current study was designed to examine this issue using a rat model of lithium-pilocarpine-induced epilepsy. Electroencephalography (EEG), immunofluorescence, and Western blot analysis showed that the loss of myelin and oligodendrocytes
Epilepsy
in the rat hippocampus began during the acute stage of epileptogenesis, and the severity of
Remyelination
this loss increased throughout epileptogenesis. Accompanying this loss of myelin and oligodendrocytes, OPCs in the rat hippocampus became activated and their populations increased during several phases of epileptogenesis (the acute, latent and chronic phases). The transcription factors olig1 and olig2, which play crucial roles in regulating OPC proliferation, differentiation and remyelination, were up-regulated during the early phases (the acute and latent phases) followed by a sharp decline in their expression during the chronic and late chronic phases. This study is the first to confirm the loss of myelin and oligodendrocytes during lithium-pilocarpine-induced epileptogenesis accompanied by a transient increase in the number of OPCs. Prevention of the loss of myelin and oligodendrocytes may provide a novel treatment strategy for epilepsy. & 2015 Published by Elsevier B.V.
Abbreviations: TLE, epileptic drugs; SC,
temporal lobe epilepsy; CNS,
status convulsion; NG2,
central nervous system; OPC,
oligodendrocyte precursor cell; AEDs, anti-
chondroitin sulfate proteoglycan; PDGFR-α,
platelet-derived growth factor receptor α;
SRS, spontaneous recurrent seizures; MBP, myelin basic protein; EEG, electroencephalography; PBS, phosphate-buffered saline n Corresponding author at: Department of Neurology, Children's Hospital of Chongqing Medical University, 136# Zhongshan 2 Road, Chongqing 400014, PR China. Fax: þ86 2363622754. E-mail address: [email protected] (L. Jiang). http://dx.doi.org/10.1016/j.brainres.2015.09.027 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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1.
Introduction
Epilepsy, a prevalent neurological disorder that is characterized by recurrent unprovoked seizures, affects more than 50 million people worldwide (Zhu et al., 2014). Previous research into the underlying pathology of epilepsy has been confined to neuronal loss, neuronal dysfunction and aberrant neuronal network function. Despite remarkable advances in epilepsy research, its underlying pathogenesis remains incompletely understood. The currently available antiepileptic drugs (AEDs), which act on various molecular targets to reduce the hyperexcitability of neurons, have resulted in great progress in controlling epileptic seizures. Nonetheless, nearly 30% of epilepsy cases are refractory to even the best available AEDs (Amini et al., 2014). Therefore, in addition to neurons, the pathogenesis of epilepsy must be explored in other cell types and structures within the central nervous system (CNS). The myelin sheath, an important structure in the CNS, is formed by mature oligodendrocytes that surround axons. The myelin sheath is critical for interneuronal communication, and the integrity of myelin is crucial for maintaining normal neuronal network function and sequential electrical impulse conduction. Although the myelin sheath has traditionally not been considered critical to the pathogenesis of epilepsy, recent reports have demonstrated that CNS myelin sheaths are affected in certain epilepsy patients (Concha et al., 2009; Nilsson et al., 2008; Scanlon et al., 2013; Schoene-Bake et al., 2009), and our previous study confirmed that demyelination of the hippocampus occurs in lithium-pilocarpine-induced epileptic rats (Ye et al., 2013). In addition, epileptic seizures have also been observed in patients with multiple sclerosis, a typical demyelinating disease of the CNS, and in different animal models with myelin diseases (Anderson and Rodriguez, 2011; Bloom et al., 2002; Eguibar and Cortes Mdel, 2010). These findings indicate an association between epilepsy and abnormal myelin damage. However, limited data are available regarding the dynamic changes to the myelin sheath and to mature oligodendrocytes that occur in the hippocampus during epileptogenesis. Oligodendrocyte precursor cells (OPCs), a type of glial cell, are capable of generating myelinating oligodendrocytes during brain development and adulthood (Levine et al., 2001). The primary function of OPCs is to serve as a reservoir of new oligodendrocytes throughout life. In experimental models of demyelination, endogenous OPCs are believed to be the primary source of new oligodendrocytes. In response to demyelination, OPCs rapidly proliferate, migrate, and finally differentiate into mature oligodendrocytes to wrap denuded axons in demyelinated areas, thereby restoring myelin sheaths (Carroll et al., 1998; Di Bello et al., 1999; Franklin, 2002; Franklin and Ffrench-Constant, 2008; Keirstead et al., 1998). The endogenous myelin repair process, remyelination, is primarily mediated by mature oligodendrocytes and OPCs. Demyelination in the hippocampus has been demonstrated in epileptic rats (Ye et al., 2013; You et al., 2011), and OPCs have the potential to regenerate mature
oligodendrocytes under demyelinating conditions. Thus, the myelin sheath, mature oligodendrocytes and OPCs are most likely involved in epileptogenesis. To test this hypothesis, we adopted a lithium-pilocarpine-induced rat model of epilepsy that reproduces most of the clinical and neuropathological features of human temporal lobe epilepsy (TLE), a form of epilepsy that is especially refractory to drug treatment (Curia et al., 2008). The natural history of epilepsy after the initial status convulsion (SC) induced by lithium–pilocarpine reproduces the process of epilepsy development in humans. Using EEG, immunofluorescence, and Western blotting, we detected dynamic changes in the myelin sheath, mature oligodendrocytes, OPCs, and related regulators in the hippocampus during epileptogenesis to identify their possible roles and relationships in this animal model.
2.
Results
2.1. Epilepsy induction and a description of the animals used in this study SC was successfully induced in 75 rats after pilocarpine administration, and the SC was interrupted after 90 min by the intraperitoneal injection of the anticonvulsant diazepam. Rats that did not exhibit SC (n ¼15) or that died from SC (n¼ 7) were excluded. The surviving rats experienced occasional, self-limiting generalized seizures (not more than one min in duration) during the acute phase of epileptogenesis after SC. When entering the latent phase (five days after SC), all rats Q4 displayed normal behavior and activity (Fig. 1). To evaluate subsequent spontaneous recurrent seizures (SRS), the behavior and cortical EEGs of the rats were monitored and evaluated by trained observers. The first SRS occurred 1172.2 days after SC in 64 of the 68 rats (94.1%). The four rats that did not display SRS after 80 days were excluded. Similarly to previously reported findings (Goffin et al., 2007; Ye et al., 2013), SRS tended to occur in clusters with a duration of less than one min for each seizure. Fig. 2 shows the number of spontaneous seizures per day during the late chronic phase after the first spontaneous seizure.
2.2. Myelin damage in rats subjected to lithiumpilocarpine-induced epileptogenesis Because myelin basic protein (MBP) is one of the major proteins in compact myelin (Vincze et al., 2008), Western blotting was performed to detect MBP expression and to evaluate the damage incurred by myelin sheaths. As shown in Fig. 3, the hippocampal expression of MBP in the epileptic group was lower than that in the control group during all phases of epileptogenesis; in addition, a significant difference in the MBP/actin expression ratio in the hippocampus was observed during multiple phases of epileptogenesis, indicating that myelin sheath damage was initiated during the acute stage of epileptogenesis and that the severity of this damage increased throughout lithium-pilocarpine-induced epileptogenesis.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 1 – Electroencephalographic recordings during epileptogenesis after SC. (A) Electroencephalographic recording in normal rats. (a) Amplification of the region labeled in (A). (B) Electroencephalographic recording of status convulsion. (b): Amplification of the region labeled in (B). (C) Electroencephalographic recording of the latent stage of epileptogenesis. (c): Amplification of the region labeled in (C). (D) Electroencephalographic recording of spontaneous recurrent seizures. (d): Amplification of the region labeled in (D).
2.3. Oligodendrocytes in rats subjected to lithiumpilocarpine-induced epileptogenesis The myelin sheath is formed by mature oligodendrocytes surrounding axons. To observe the dynamic changes occurring in mature oligodendrocytes during epileptogenesis, immunohistochemistry was used to study the expression of CC1, a marker of mature oligodendrocytes (Flygt et al., 2013; Sun et al., 2010). Fig. 4B shows the numbers of CC1-positive hippocampal mature oligodendrocytes in different groups. A one-way ANOVA revealed significant differences in the numbers of CC1-positive oligodendrocytes between the epileptic and control groups. In accordance with the myelin changes, the number of CC1-positive oligodendrocytes began to decrease during the acute phase of epileptogenesis, and the severity of this loss lasted throughout lithium-pilocarpineinduced epileptogenesis.
2.4. Oligodendrocyte precursor cells in rats subjected to lithium-pilocarpine-induced epileptogenesis
Fig. 2 – Number of spontaneous seizures per day (y-axis) after the first spontaneous seizure.
We sought to determine the dynamic changes in OPCs that occur during epileptogenesis. Immunohistochemistry and Western blotting were used to study the expression of chondroitin sulfate proteoglycan (NG2) and platelet-derived growth factor receptor α (PDGFR-α) in the rat hippocampus.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Both PDGFR-α and NG2 have been found to be specifically expressed in OPCs and have been widely used as markers of OPCs (He et al., 2009; Horner et al., 2002; Wilson et al., 2006). As shown in Fig. 5A, double immunofluorescence staining for PDGFR-α and NG2 showed that these two proteins were colocalized with a population of stellate-shaped cells in the rat hippocampus. Compared with PDGFR-α, NG2 more clearly showed the morphology of OPCs. Therefore, to study the number and morphology of OPCs, sections of rat brains from different groups were labeled with NG2 antibodies. The NG2positive OPCs in the hippocampus of the control group had small, round cell bodies and multi-branched processes radiating in all directions. After SC, hippocampal NG2positive OPCs showed an increase in the sizes of their cell bodies and processes, as well as increased staining intensity. These cells were characterized as reactive OPCs, also called activated OPCs (Fig. 6A). These reactive OPCs were found in the hippocampus immediately after SC during the acute phase of epileptogenesis and remained present during the chronic phase but were absent during the late chronic phase. Ki67 is a marker of endogenous proliferation. Double immunofluorescence staining for NG2 and Ki67 revealed that a subset of these reactive OPCs expressed Ki67, indicating that these OPCs were dividing cells (Fig. 6A).
Fig. 5B shows the numbers of NG2-positive hippocampal OPCs in different groups. A one-way ANOVA revealed significant differences in the numbers of NG2-positive OPCs between the epileptic and control groups. The number of NG2-positive OPCs began to increase during the acute phase of epileptogenesis, peaked during the latent phase, and then decreased during the chronic phase. However, during the late chronic phase, the number of NG2-positive OPCs in the epileptic group was lower than that in the control group. To further explore changes in the ability of OPCs to proliferate, the percentage of Ki67-positive OPCs was analyzed in the same regions in which we had previously quantified OPCs (Fig. 6B). As shown in Fig. 6B, in accordance with the changes in the numbers of NG2-positive OPCs, the percentage of Ki67positive OPCs began to increase immediately following SC in the acute phase, peaked during the latent phase, and then decreased during the chronic phase. The percentage of Ki67positive OPCs in the experimental group was lower than that in the control group during the late chronic phase. This phenomenon revealed that the ability of OPCs to proliferate began to increase during the acute phase, peaked during the latent phase, decreased during the chronic phase, and then decreased below that of the control group during the late chronic phase. To determine the presence of changes in OPCs at the protein level, Western blotting was used to detect the protein expression levels of PDGFR-α (Fig. 3). Consistent with the immunohistochemical observation of NG2-positive OPCs, Fig. 3 – Protein expression levels of MBP, PDGFR-α, olig1 and olig2 in the hippocampi of rats in the experimental groups (acute phase, latent phase, chronic phase, and late chronic phase groups) and in the control group were detected via Western blot. The image shows representative immunoblots of MBP, PDGFR-α, olig1 and olig2 proteins in the hippocampus; (A) MBP and PDGFR-α levels were normalized to those of βactin in the hippocampus. The MBP/β-actin and PDGFR-α/βactin expression ratios are presented as the relative optical densities. As shown in the image, the level of MBP gradually decreased throughout epileptogenesis. The levels of PDGFR-α protein in the hippocampus immediately increased during the acute phase of epileptogenesis and peaked during the latent phase. Over time, the levels of PDGFR-α protein began to decrease during the chronic phase; ultimately, the levels of PDGFR-α protein were lower in the experimental group than in the control group during the late chronic phase. Statistical significance: *po0.01 versus the normal control group; ▲ po0.01 versus the former group. (B) Olig1 and olig2 levels were normalized to those of β-actin in the hippocampus. The olig1/β-actin and olig2/β-actin expression ratios are presented as the relative optical densities. The protein levels of olig1 and olig2 in the hippocampus immediately increased during the acute phase of epileptogenesis and peaked during the latent phase. Sharp declines in the protein levels of olig1 and olig2 were then observed during the chronic and late chronic phases, and their expression levels were significantly lower in the experimental group than in the control group during both the chronic and late chronic phases. Statistical significance: *po0.01 versus the normal control group; S po0.01 versus the former group.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 4 – (A) Representative images of immunofluorescence staining for CC1 in the CA1 region of rat hippocampi in the experimental groups (acute phase, latent phase, chronic phase, and late chronic phase groups) and in the control group. Green fluorescence indicates CC1 protein, and blue fluorescence indicates the nuclear marker DAPI; scale bar: 50 lm. (B) The number of CC1-positive cells was detected via immunofluorescence in the hippocampi of the experimental and control groups. Compared with the normal control group, as shown in the image, the number of CC1-positive cells began to decrease during the acute phase of epileptogenesis, and the severity of this loss lasted throughout lithium-pilocarpine-induced epileptogenesis. *po0.01 versus the normal control group; ppo0.01 versus the former group. Western blot analysis showed a significant increase in PDGFR-α protein expression during the acute phase of epileptogenesis in the epileptic group compared with the control group; PDGFR-α protein expression peaked during the latent
phase and then decreased slightly during the chronic phase. However, during the late chronic phase, the protein expression of PDGFR-α was significantly lower in the epileptic groups than in the control group.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 5 – (A) Representative images of double immunofluorescence staining for PDGFR-α and NG2 in the CA1 region of the rat hippocampus. The red arrows indicate NG2-positive cells; the yellow arrows indicate PDGFR-α-positive cells; and the white arrows indicate cells co-expressing these markers. As shown in the image, all NG2-positive cells co-expressed PDGFR-α. Scale bar: 100 lm. (B) The number of NG2-positive cells was detected via immunofluorescence in the hippocampi of animals from the experimental groups (acute phase, latent phase, chronic phase, and late chronic phase groups) and the control group. Compared with the normal control group, the experimental groups displayed increased numbers of NG2-positive cells in the hippocampus immediately after SC during the acute phase. The number of NG2-positive cells peaked during the latent phase and then decreased during the chronic phase. Fewer NG2-positive cells were observed in the experimental group than in the control group during the late chronic phase (*po0.05 versus the normal control group).
2.5. Olig1 and olig2 in rats subjected to lithiumpilocarpine-induced epileptogenesis Both olig1 and olig2 are basic helix-loop-helix (bHLH) transcription factors that play crucial roles in the regulation of OPC proliferation, differentiation and remyelination. To determine whether transcription factors that regulate the proliferation, differentiation and remyelination of OPCs participate in epileptogenesis, the protein expression levels of olig1 and olig2 in the rat hippocampus were detected via Western blot. Fig. 3 shows that the expression levels of olig1 and olig2 were up-regulated during the acute phase of
epileptogenesis and peaked during the latent phase. The expression of these proteins then declined sharply during the chronic and late chronic phases, and their expression was significantly lower in the epileptic group than in the control group during both the chronic and late chronic phases.
3.
Discussion
Epilepsy is a prevalent neurological disorder that is characterized by recurrent unprovoked seizures, which are caused by instant abnormal hypersynchronous electrical activity in a
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 6 – (A) Representative images of double immunofluorescence labeling for NG2 and the endogenous proliferation marker Ki67 in the hippocampi of rats in the control group (A1–A3) and the experimental group (B1–B3). The red arrows indicate NG2positive cells, the yellow arrows indicate Ki67-positive cells, and the white arrows indicate cells co-expressing both markers, representing dividing NG2-positive cells. As shown in the image, the NG2-positive cells in the experimental group exhibited a reactive status, which was characterized by enlarged cell bodies, numerous membrane blebs and many short filopodial-like processes. Scale bar: 100 lm. (B) The percentage of Ki67-positive OPCs was analyzed in the hippocampi of animals from the experimental groups and the control group. As in (B), the percentage of Ki67-positive OPCs began to increase immediately following SC in the acute phase, peaked during the latent phase, and then decreased during the chronic phase. Finally, the percentage of Ki67-positive OPCs in the experimental group was lower than that in the control group during the late chronic phase. This phenomenon indicates that the ability of OPCs to proliferate began to increase during the acute phase, peaked during the latent phase, and then decreased during the chronic phase.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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neural network (Fisher et al., 2005). Previous studies of the underlying pathology of epilepsy have confirmed that myelin is affected in certain patients with epilepsy and in epileptic rats (Ye et al., 2013; You et al., 2011), indicating an association between epilepsy and myelin damage. The present study revealed that the myelin damage observed in the rat hippocampus began during the acute stage of lithium-pilocarpine-induced epileptogenesis and that the severity of this damage increased throughout the natural trajectory of epilepsy, leading to an abundance of demyelination. To further explore the mechanism resulting in an increase in myelin damage, changes in oligodendrocytes, which are myelin-forming cells, were detected. Our results showed that accompanying this myelin damage, the number of CC1-positive oligodendrocytes decreased throughout all phases of lithium-pilocarpine-induced epileptogenesis, indicating that the myelin damage is partly attributable to the ongoing death of CC1-positive oligodendrocytes. Because of the crucial role of myelin and oligodendrocytes in protecting and supporting the axon and because the integrity of myelin is crucial for maintaining normal neuronal network function and sequential electrical impulse conduction (Piaton et al., 2010), the loss of myelin and oligodendrocytes may contribute to the pathology of epileptogenesis. OPCs, which comprise approximately 5–8% of the total glial population in the adult CNS, are capable of generating myelinating oligodendrocytes during both brain development and adulthood (Levine et al., 2001; Polito and Reynolds, 2005). Although the function of OPCs is incompletely understood, one of their precise functions is to serve as a reservoir of new oligodendrocytes throughout life (Polito and Reynolds, 2005). In the normal adult brain, OPCs remain in a quiescent state in which these cells divide infrequently (Dawson et al., 2003). Upon myelin damage, such as that which occurs in experimental demyelination models, OPCs are rapidly recruited to the traumatized areas and ultimately differentiate into mature oligodendrocytes to restore myelin sheaths (Di Bello et al., 1999; Levine and Reynolds, 1999; Watanabe et al., 2002). The endogenous myelin repair process, termed remyelination, is primarily mediated by oligodendrocytes and OPCs. Based on immunofluorescence using NG2 and Ki67 antibodies, we also demonstrated that NG2-positive OPCs in the rat hippocampus underwent increased NG2 expression and morphological changes, such as enlargement of the soma and processes, which were observed during several phases (the acute, latent, and chronic phases) of epileptogenesis. Increased numbers of OPCs and an increased percentage of Ki67-positive OPCs were observed to be concurrent with these morphological changes, indicating that OPCs rapidly activated and proliferated after SC induced by lithium–pilocarpine. We can speculate on the possible implications of the changes observed in OPCs during the process of lithiumpilocarpine-induced epileptogenesis. Similar morphological changes in NG2-positive OPCs have been reported in some diseases that exhibit white matter injury, such as mechanical brain injury (Levine, 1994), focal ischemia (Tanaka et al., 2001), and experimental autoimmune encephalomyelitis (Nishiyama et al., 1997). These changes, including altered morphology, enhanced proliferation and increased NG2 protein expression, are generally regarded as indicators of the
activation of NG2-positive OPCs (Levine, 1994; Levine and Reynolds, 1999; Nishiyama et al., 1997). Because OPCs have the capacity to mature into myelinating oligodendrocytes for remyelination, the transition from a quiescent to an activated state may reflect an attempt to compensate for the loss of mature oligodendrocytes. Considering that this transition concurrently occurred with demyelination and oligodendrocyte loss during this experiment, we speculate that the activation of OPCs represent an attempt by OPCs to differentiate into mature oligodendrocytes to remyelinate damaged myelin, indicating that an endogenous repair process is initiated in the process of lithium-pilocarpine-induced epileptogenesis. However, as our results showed, the increased number of OPCS and their increased ability to proliferate were observed merely during several phases of epileptogenesis (the acute, latent, and chronic phases), indicating the activation of OPCs is just a transient process. In addition, despite the increased number of OPCs observed during several phases of epileptogenesis (the acute, latent, and chronic phases), myelin damage and the loss of oligodendrocytes were observed throughout epileptogenesis, indicating the lack of efficient endogenous repair. To explore the possible mechanism behind the transient activation of OPCs during epileptogenesis, we detected dynamic changes in two transcription factors, olig1 and olig2, which are crucial for the proliferation and differentiation of OPCs into mature oligodendrocytes during myelination and remyelination. Olig1 and olig2 are closely related bHLH transcription factors that are expressed in oligodendroglial lineage cells in the CNS (Lu et al., 2002). Knockout mouse studies have demonstrated that olig1 is required for the generation of mature oligodendrocytes and that olig2 plays a crucial role in the initial specification of the oligodendrocyte lineage during developmental myelination (Lu et al., 2002; Xin et al., 2005). Moreover, during remyelination in the adult brain, olig1 and olig2 continue to perform an important function. Arnett et al. found that the observed decrease in remyelination is caused by the impaired ability of OPCs to differentiate in olig1-/- mice after demyelination is induced by several toxins. This observation indicates that olig1 plays a crucial role in regulating oligodendrocyte differentiation and consequent remyelination in the adult brain (Arnett et al., 2004). Fancy et al. (2004) have demonstrated that the upregulation of the olig2 protein is critical to the activation of a quiescent population of OPCs following demyelination and enables these cells to mature into myelinating oligodendrocytes to repair damaged myelin sheaths. These findings indicate the crucial roles of olig1 and olig2 in enabling OPCs to differentiate into new oligodendrocytes to remyelinate damaged myelin sheaths. In the present study, we found that the expression levels of olig1 and olig2 were up-regulated during the acute phase, peaked during the latent phase, and then sharply declined during the chronic phase of epileptogenesis. Based on their functions in myelination and remyelination, we speculate that the increased expression of olig1 and olig2 during the early phases (the acute and latent phases) of epileptogenesis may reflect an attempt to assist activating OPCs to compensate for the loss of myelin that occurs during epileptogenesis. The sharp decline in olig1 and olig2 expression during the
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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chronic and late chronic phases, during which spontaneous recurrent seizures (SRS) occur, may contribute to the inhibition of OPC activation, proliferation and differentiation, resulting in the restriction of the endogenous repair of myelin during epileptogenesis. However, although our findings suggest that the activation of OPCs may contribute to the endogenous repair process by supplying new oligodendrocytes, we have no direct evidence that the activated OPCs are involved in remyelination. Further study is needed to address the following issues: (1) whether the activated OPCs successfully differentiate into mature oligodendrocytes and (2) the fate (death or differentiation) of the decreased population of OPCs in chronic and late chronic phases. Overall, our findings demonstrate for the first time that loss of myelin accumulates during lithium-pilocarpineinduced epileptogenesis. Myelin damage was accompanied by the loss of oligodendrocytes, indicating that myelin damage is partly secondary to the loss of oligodendrocytes. The activation of OPCs, although transient, indicates that an endogenous repair process is initiated during epileptogenesis and that the transient activation of OPCs may be partly due to changes in its related regulatory factors. Preventing damage to the myelin sheath and promoting the proliferation and differentiation of OPCs to participate in this endogenous repair process may provide a promising treatment strategy for epilepsy.
4.
Experimental procedures
4.1.
Animals
For this study, 106 adult Sprague–Dawley rats weighing 200– 250 g (Experimental Animal Center, Chongqing Medical University, Chongqing, China) were group housed and provided with ad libitum access to water and food. The animals were housed with a 12 h light/dark cycle and at a temperature of 22–24 1C and a relative humidity of 50–60%. The Administrative Panel on Laboratory Animal Care of Chongqing Medical University approved all experimental procedures.
4.2. Preparation of the epilepsy model and video–EEG monitoring The epilepsy modeling and video–EEG monitoring were conducted as previously described (Ye et al., 2013). One week before inducing seizure convulsion, the rats were anesthetized via intraperitoneal (i.p.) injection of 10% chloral hydrate (3 ml/kg) and placed in a stereotactic frame. To record cortical EEGs, a pair of insulated stainless steel electrodes was inserted into the skull bilaterally over the parietal cortex, and a ground lead was positioned over the nasal sinus. After four days of recovery, the rats were monitored via continuous cortical electroencephalography (EEG). All EEG recordings and video data were visually screened, and seizures were confirmed by trained human observers. For the preparation of the epilepsy animal model, SC was induced in the rats four days after surgery via i.p. injection of pilocarpine (pilo, 50 mg/kg, Sigma) 18 h after the injection of
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lithium (127 mg/kg, i.p, Solarbio, Beijing, China). To reduce peripheral effects, methyl-scopolamine was administered via an i.p. injection (1 mg/kg) 30 min prior to the pilo injection. Ninety minutes after SC, all rats received a single dose of diazepam (10 mg/kg, i.p.) The rats in the control group received an equivalent volume of 9% saline. To evaluate the dynamic changes in myelin and OPCs, the rats were sacrificed at various time points after SC reflecting the different phases of the natural trajectory of epilepsy: the acute phase of epileptogenesis was defined as the first 24 h after acute SC; the latent phase consisted of the following five days; the chronic phase was defined as the two weeks after the first spontaneous recurrent seizure (SRS); and the late chronic phase was defined as the two months after the first SRS (Mazzuferi et al., 2010). According to previous descriptions, the threshold discharge for the first spontaneous electrographic seizure was set at a frequency of 45 Hz, an amplitude of 42 baseline and a duration of 410 s (Goffin et al., 2007). The number of spontaneous seizures in the rats during the late chronic phase was monitored for one month after the first spontaneous seizure. Rats that did not exhibit SC, that died, or that did not develop spontaneous seizures were excluded.
4.3.
Immunofluorescence
The rats (8 animals per group) were anesthetized via an i.p. injection of 10% chloral hydrate (3 ml/kg) and were then transcardially perfused with 120 ml of 9% saline followed by 250 ml of 4% paraformaldehyde. After perfusion, each brain was removed and placed in 4% paraformaldehyde containing 30% sucrose at 4 1C until the brain sank. Sagittal sections were serially cut at a thickness of 40 μm using a freezing microtome and were stored in cryoprotectant solution at 4 1C until immunofluorescence staining was performed. The following primary antibodies/combinations of antibodies were used: NG2 (mouse monoclonal, Chemicon) and PDGFR-α (rabbit polyclonal, Santa Cruz Biotech), each at 1:200; CC1 (mouse monoclonal, Chemicon) at 1:200; Ki67 (rabbit polyclonal, Chemicon) and NG2 (same as above) at 1:1000 and 1:200, respectively; or NG2 alone (same as above) at 1:200. All of the antibodies listed were incubated with tissue overnight. All images were captured using a laser-scanning confocal microscope.
4.4.
Western blotting
The hippocampus of each rat (8 animals per group) was separately collected at the indicated phase of epileptogenesis (acute, latent, chronic, or late chronic phase) and stored in liquid nitrogen immediately after dissection. Total protein samples were extracted using a protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer's instructions, and the protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, USA). Equal amounts of protein (30 μg per lane) were loaded on 8–16% SDS polyacrylamide gels. After electrophoresis, the samples were transferred to polyvinylidene difluoride membranes (0.22 μm, Millipore Corp., Billerica, MA, USA) and the membranes were blocked for 1 h in PBS containing 10% skim
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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milk. The membranes were further incubated in primary antibodies overnight at 4 1C followed by incubation in peroxidase-conjugated secondary antibodies. The primary antibodies used were as follows: MBP (SMI99, Covance, Emeryville, CA, USA) at 1:2000; PDGFR-α (rabbit polyclonal, Santa Cruz Biotech) at 1:1000; olig1 (mouse monoclonal, Chemicon) at 1:1000; and olig2 (mouse monoclonal, Chemicon). After 35-min washes with PBS containing 0.1% Tween20 (PBS-T) on a shaker, the membranes were processed using enhanced chemiluminescence. To normalize the results, parallel Western blots probed with an anti-β-actin antibody (1:1000, Chemicon) were developed. The Western blot bands were analyzed via densitometry using ACDsee pro 2.5 and Image J software.
4.5.
Data collection and statistical analysis
EEG analysis was performed as previously described; additionally, discharges with frequencies of 45 Hz, amplitudes of 42 baseline, and durations of 410 s were regarded as spontaneous seizures (Goffin et al., 2007; Ye et al., 2013). For image analysis, the numbers of NG2-positive cells and CC1-positive cells were counted within uniform areas (200 microscopic fields) from various hippocampal regions (CA1, CA3, and DG). The numbers of NG2-positive cells, Ki67- and NG2- double positive cells and CC1-positive cells from at least five consecutive coronal sections in each animal (n¼ 8) were averaged. All data are presented as the means7SD and were analyzed via one-way ANOVA followed by either Dunnett's or Tukey's post hoc test to determine the statistical significance of the observed difference; po0.05 was considered statistically significant.
Author contributions Yuanyuan Luo and Li Jiang conceived and designed the experiments. Yuanyuan Luo performed the experiments and analyzed the data. Qiao Hu, Qian Zhang, Siqi Hong, Xiaoju Tang, Zhengxiong Yao, and Li Chen contributed reagents, materials, and analysis tools. Yuanyuan Luo wrote the manuscript.
Acknowledgments Q5 This work was supported by the National Natural Science
Foundation of China (NSFC Grant 81371452), the National Natural Science Foundation of Chongqing (CSCT 2013jjB0031), and the Research Fund for the Doctoral Program of Higher Education of China (20125503110011).
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Research Report
Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis Q1
Yuanyuan Luoa, Qiao Hua, Qian Zhanga, Siqi Honga,b, Xiaoju Tanga, Li Chena, Li Jianga,b,n a Lab of Pediatric Neurology, Ministry of Education, Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Children’s Hospital of Chongqing Medical University, Chongqing, PR China b Department of Neurology, Children’s Hospital of Chongqing Medical University, 136# Zhongshan 2 Road, Chongqing 400014, PR China
art i cle i nfo
ab st rac t
Article history:
Recent reports have described damage to myelinated fibers in the central nervous system
Accepted 22 September 2015
(CNS) in patients with temporal lobe epilepsy (TLE) and animal models. However, only limited data are available on the dynamic changes that occur in myelinated fibers, oligodendrocytes (which are myelin-forming cells), and oligodendrocyte precursor cells
Keywords:
(OPCs), which are a reservoir of new oligodendrocytes, in the hippocampus throughout
Myelin sheath Oligodendrocyte precursor cells Oligodendrocyte
epileptogenesis. The current study was designed to examine this issue using a rat model of lithium-pilocarpine-induced epilepsy. Electroencephalography (EEG), immunofluorescence, and Western blot analysis showed that the loss of myelin and oligodendrocytes
Epilepsy
in the rat hippocampus began during the acute stage of epileptogenesis, and the severity of
Remyelination
this loss increased throughout epileptogenesis. Accompanying this loss of myelin and oligodendrocytes, OPCs in the rat hippocampus became activated and their populations increased during several phases of epileptogenesis (the acute, latent and chronic phases). The transcription factors olig1 and olig2, which play crucial roles in regulating OPC proliferation, differentiation and remyelination, were up-regulated during the early phases (the acute and latent phases) followed by a sharp decline in their expression during the chronic and late chronic phases. This study is the first to confirm the loss of myelin and oligodendrocytes during lithium-pilocarpine-induced epileptogenesis accompanied by a transient increase in the number of OPCs. Prevention of the loss of myelin and oligodendrocytes may provide a novel treatment strategy for epilepsy. & 2015 Published by Elsevier B.V.
Abbreviations: TLE, epileptic drugs; SC,
temporal lobe epilepsy; CNS,
status convulsion; NG2,
central nervous system; OPC,
oligodendrocyte precursor cell; AEDs, anti-
chondroitin sulfate proteoglycan; PDGFR-α,
platelet-derived growth factor receptor α;
SRS, spontaneous recurrent seizures; MBP, myelin basic protein; EEG, electroencephalography; PBS, phosphate-buffered saline n Corresponding author at: Department of Neurology, Children's Hospital of Chongqing Medical University, 136# Zhongshan 2 Road, Chongqing 400014, PR China. Fax: þ86 2363622754. E-mail address: [email protected] (L. Jiang). http://dx.doi.org/10.1016/j.brainres.2015.09.027 0006-8993/& 2015 Published by Elsevier B.V.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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1.
Introduction
Epilepsy, a prevalent neurological disorder that is characterized by recurrent unprovoked seizures, affects more than 50 million people worldwide (Zhu et al., 2014). Previous research into the underlying pathology of epilepsy has been confined to neuronal loss, neuronal dysfunction and aberrant neuronal network function. Despite remarkable advances in epilepsy research, its underlying pathogenesis remains incompletely understood. The currently available antiepileptic drugs (AEDs), which act on various molecular targets to reduce the hyperexcitability of neurons, have resulted in great progress in controlling epileptic seizures. Nonetheless, nearly 30% of epilepsy cases are refractory to even the best available AEDs (Amini et al., 2014). Therefore, in addition to neurons, the pathogenesis of epilepsy must be explored in other cell types and structures within the central nervous system (CNS). The myelin sheath, an important structure in the CNS, is formed by mature oligodendrocytes that surround axons. The myelin sheath is critical for interneuronal communication, and the integrity of myelin is crucial for maintaining normal neuronal network function and sequential electrical impulse conduction. Although the myelin sheath has traditionally not been considered critical to the pathogenesis of epilepsy, recent reports have demonstrated that CNS myelin sheaths are affected in certain epilepsy patients (Concha et al., 2009; Nilsson et al., 2008; Scanlon et al., 2013; Schoene-Bake et al., 2009), and our previous study confirmed that demyelination of the hippocampus occurs in lithium-pilocarpine-induced epileptic rats (Ye et al., 2013). In addition, epileptic seizures have also been observed in patients with multiple sclerosis, a typical demyelinating disease of the CNS, and in different animal models with myelin diseases (Anderson and Rodriguez, 2011; Bloom et al., 2002; Eguibar and Cortes Mdel, 2010). These findings indicate an association between epilepsy and abnormal myelin damage. However, limited data are available regarding the dynamic changes to the myelin sheath and to mature oligodendrocytes that occur in the hippocampus during epileptogenesis. Oligodendrocyte precursor cells (OPCs), a type of glial cell, are capable of generating myelinating oligodendrocytes during brain development and adulthood (Levine et al., 2001). The primary function of OPCs is to serve as a reservoir of new oligodendrocytes throughout life. In experimental models of demyelination, endogenous OPCs are believed to be the primary source of new oligodendrocytes. In response to demyelination, OPCs rapidly proliferate, migrate, and finally differentiate into mature oligodendrocytes to wrap denuded axons in demyelinated areas, thereby restoring myelin sheaths (Carroll et al., 1998; Di Bello et al., 1999; Franklin, 2002; Franklin and Ffrench-Constant, 2008; Keirstead et al., 1998). The endogenous myelin repair process, remyelination, is primarily mediated by mature oligodendrocytes and OPCs. Demyelination in the hippocampus has been demonstrated in epileptic rats (Ye et al., 2013; You et al., 2011), and OPCs have the potential to regenerate mature
oligodendrocytes under demyelinating conditions. Thus, the myelin sheath, mature oligodendrocytes and OPCs are most likely involved in epileptogenesis. To test this hypothesis, we adopted a lithium-pilocarpine-induced rat model of epilepsy that reproduces most of the clinical and neuropathological features of human temporal lobe epilepsy (TLE), a form of epilepsy that is especially refractory to drug treatment (Curia et al., 2008). The natural history of epilepsy after the initial status convulsion (SC) induced by lithium–pilocarpine reproduces the process of epilepsy development in humans. Using EEG, immunofluorescence, and Western blotting, we detected dynamic changes in the myelin sheath, mature oligodendrocytes, OPCs, and related regulators in the hippocampus during epileptogenesis to identify their possible roles and relationships in this animal model.
2.
Results
2.1. Epilepsy induction and a description of the animals used in this study SC was successfully induced in 75 rats after pilocarpine administration, and the SC was interrupted after 90 min by the intraperitoneal injection of the anticonvulsant diazepam. Rats that did not exhibit SC (n ¼15) or that died from SC (n¼ 7) were excluded. The surviving rats experienced occasional, self-limiting generalized seizures (not more than one min in duration) during the acute phase of epileptogenesis after SC. When entering the latent phase (five days after SC), all rats Q4 displayed normal behavior and activity (Fig. 1). To evaluate subsequent spontaneous recurrent seizures (SRS), the behavior and cortical EEGs of the rats were monitored and evaluated by trained observers. The first SRS occurred 1172.2 days after SC in 64 of the 68 rats (94.1%). The four rats that did not display SRS after 80 days were excluded. Similarly to previously reported findings (Goffin et al., 2007; Ye et al., 2013), SRS tended to occur in clusters with a duration of less than one min for each seizure. Fig. 2 shows the number of spontaneous seizures per day during the late chronic phase after the first spontaneous seizure.
2.2. Myelin damage in rats subjected to lithiumpilocarpine-induced epileptogenesis Because myelin basic protein (MBP) is one of the major proteins in compact myelin (Vincze et al., 2008), Western blotting was performed to detect MBP expression and to evaluate the damage incurred by myelin sheaths. As shown in Fig. 3, the hippocampal expression of MBP in the epileptic group was lower than that in the control group during all phases of epileptogenesis; in addition, a significant difference in the MBP/actin expression ratio in the hippocampus was observed during multiple phases of epileptogenesis, indicating that myelin sheath damage was initiated during the acute stage of epileptogenesis and that the severity of this damage increased throughout lithium-pilocarpine-induced epileptogenesis.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 1 – Electroencephalographic recordings during epileptogenesis after SC. (A) Electroencephalographic recording in normal rats. (a) Amplification of the region labeled in (A). (B) Electroencephalographic recording of status convulsion. (b): Amplification of the region labeled in (B). (C) Electroencephalographic recording of the latent stage of epileptogenesis. (c): Amplification of the region labeled in (C). (D) Electroencephalographic recording of spontaneous recurrent seizures. (d): Amplification of the region labeled in (D).
2.3. Oligodendrocytes in rats subjected to lithiumpilocarpine-induced epileptogenesis The myelin sheath is formed by mature oligodendrocytes surrounding axons. To observe the dynamic changes occurring in mature oligodendrocytes during epileptogenesis, immunohistochemistry was used to study the expression of CC1, a marker of mature oligodendrocytes (Flygt et al., 2013; Sun et al., 2010). Fig. 4B shows the numbers of CC1-positive hippocampal mature oligodendrocytes in different groups. A one-way ANOVA revealed significant differences in the numbers of CC1-positive oligodendrocytes between the epileptic and control groups. In accordance with the myelin changes, the number of CC1-positive oligodendrocytes began to decrease during the acute phase of epileptogenesis, and the severity of this loss lasted throughout lithium-pilocarpineinduced epileptogenesis.
2.4. Oligodendrocyte precursor cells in rats subjected to lithium-pilocarpine-induced epileptogenesis
Fig. 2 – Number of spontaneous seizures per day (y-axis) after the first spontaneous seizure.
We sought to determine the dynamic changes in OPCs that occur during epileptogenesis. Immunohistochemistry and Western blotting were used to study the expression of chondroitin sulfate proteoglycan (NG2) and platelet-derived growth factor receptor α (PDGFR-α) in the rat hippocampus.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Both PDGFR-α and NG2 have been found to be specifically expressed in OPCs and have been widely used as markers of OPCs (He et al., 2009; Horner et al., 2002; Wilson et al., 2006). As shown in Fig. 5A, double immunofluorescence staining for PDGFR-α and NG2 showed that these two proteins were colocalized with a population of stellate-shaped cells in the rat hippocampus. Compared with PDGFR-α, NG2 more clearly showed the morphology of OPCs. Therefore, to study the number and morphology of OPCs, sections of rat brains from different groups were labeled with NG2 antibodies. The NG2positive OPCs in the hippocampus of the control group had small, round cell bodies and multi-branched processes radiating in all directions. After SC, hippocampal NG2positive OPCs showed an increase in the sizes of their cell bodies and processes, as well as increased staining intensity. These cells were characterized as reactive OPCs, also called activated OPCs (Fig. 6A). These reactive OPCs were found in the hippocampus immediately after SC during the acute phase of epileptogenesis and remained present during the chronic phase but were absent during the late chronic phase. Ki67 is a marker of endogenous proliferation. Double immunofluorescence staining for NG2 and Ki67 revealed that a subset of these reactive OPCs expressed Ki67, indicating that these OPCs were dividing cells (Fig. 6A).
Fig. 5B shows the numbers of NG2-positive hippocampal OPCs in different groups. A one-way ANOVA revealed significant differences in the numbers of NG2-positive OPCs between the epileptic and control groups. The number of NG2-positive OPCs began to increase during the acute phase of epileptogenesis, peaked during the latent phase, and then decreased during the chronic phase. However, during the late chronic phase, the number of NG2-positive OPCs in the epileptic group was lower than that in the control group. To further explore changes in the ability of OPCs to proliferate, the percentage of Ki67-positive OPCs was analyzed in the same regions in which we had previously quantified OPCs (Fig. 6B). As shown in Fig. 6B, in accordance with the changes in the numbers of NG2-positive OPCs, the percentage of Ki67positive OPCs began to increase immediately following SC in the acute phase, peaked during the latent phase, and then decreased during the chronic phase. The percentage of Ki67positive OPCs in the experimental group was lower than that in the control group during the late chronic phase. This phenomenon revealed that the ability of OPCs to proliferate began to increase during the acute phase, peaked during the latent phase, decreased during the chronic phase, and then decreased below that of the control group during the late chronic phase. To determine the presence of changes in OPCs at the protein level, Western blotting was used to detect the protein expression levels of PDGFR-α (Fig. 3). Consistent with the immunohistochemical observation of NG2-positive OPCs, Fig. 3 – Protein expression levels of MBP, PDGFR-α, olig1 and olig2 in the hippocampi of rats in the experimental groups (acute phase, latent phase, chronic phase, and late chronic phase groups) and in the control group were detected via Western blot. The image shows representative immunoblots of MBP, PDGFR-α, olig1 and olig2 proteins in the hippocampus; (A) MBP and PDGFR-α levels were normalized to those of βactin in the hippocampus. The MBP/β-actin and PDGFR-α/βactin expression ratios are presented as the relative optical densities. As shown in the image, the level of MBP gradually decreased throughout epileptogenesis. The levels of PDGFR-α protein in the hippocampus immediately increased during the acute phase of epileptogenesis and peaked during the latent phase. Over time, the levels of PDGFR-α protein began to decrease during the chronic phase; ultimately, the levels of PDGFR-α protein were lower in the experimental group than in the control group during the late chronic phase. Statistical significance: *po0.01 versus the normal control group; ▲ po0.01 versus the former group. (B) Olig1 and olig2 levels were normalized to those of β-actin in the hippocampus. The olig1/β-actin and olig2/β-actin expression ratios are presented as the relative optical densities. The protein levels of olig1 and olig2 in the hippocampus immediately increased during the acute phase of epileptogenesis and peaked during the latent phase. Sharp declines in the protein levels of olig1 and olig2 were then observed during the chronic and late chronic phases, and their expression levels were significantly lower in the experimental group than in the control group during both the chronic and late chronic phases. Statistical significance: *po0.01 versus the normal control group; S po0.01 versus the former group.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 4 – (A) Representative images of immunofluorescence staining for CC1 in the CA1 region of rat hippocampi in the experimental groups (acute phase, latent phase, chronic phase, and late chronic phase groups) and in the control group. Green fluorescence indicates CC1 protein, and blue fluorescence indicates the nuclear marker DAPI; scale bar: 50 lm. (B) The number of CC1-positive cells was detected via immunofluorescence in the hippocampi of the experimental and control groups. Compared with the normal control group, as shown in the image, the number of CC1-positive cells began to decrease during the acute phase of epileptogenesis, and the severity of this loss lasted throughout lithium-pilocarpine-induced epileptogenesis. *po0.01 versus the normal control group; ppo0.01 versus the former group. Western blot analysis showed a significant increase in PDGFR-α protein expression during the acute phase of epileptogenesis in the epileptic group compared with the control group; PDGFR-α protein expression peaked during the latent
phase and then decreased slightly during the chronic phase. However, during the late chronic phase, the protein expression of PDGFR-α was significantly lower in the epileptic groups than in the control group.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 5 – (A) Representative images of double immunofluorescence staining for PDGFR-α and NG2 in the CA1 region of the rat hippocampus. The red arrows indicate NG2-positive cells; the yellow arrows indicate PDGFR-α-positive cells; and the white arrows indicate cells co-expressing these markers. As shown in the image, all NG2-positive cells co-expressed PDGFR-α. Scale bar: 100 lm. (B) The number of NG2-positive cells was detected via immunofluorescence in the hippocampi of animals from the experimental groups (acute phase, latent phase, chronic phase, and late chronic phase groups) and the control group. Compared with the normal control group, the experimental groups displayed increased numbers of NG2-positive cells in the hippocampus immediately after SC during the acute phase. The number of NG2-positive cells peaked during the latent phase and then decreased during the chronic phase. Fewer NG2-positive cells were observed in the experimental group than in the control group during the late chronic phase (*po0.05 versus the normal control group).
2.5. Olig1 and olig2 in rats subjected to lithiumpilocarpine-induced epileptogenesis Both olig1 and olig2 are basic helix-loop-helix (bHLH) transcription factors that play crucial roles in the regulation of OPC proliferation, differentiation and remyelination. To determine whether transcription factors that regulate the proliferation, differentiation and remyelination of OPCs participate in epileptogenesis, the protein expression levels of olig1 and olig2 in the rat hippocampus were detected via Western blot. Fig. 3 shows that the expression levels of olig1 and olig2 were up-regulated during the acute phase of
epileptogenesis and peaked during the latent phase. The expression of these proteins then declined sharply during the chronic and late chronic phases, and their expression was significantly lower in the epileptic group than in the control group during both the chronic and late chronic phases.
3.
Discussion
Epilepsy is a prevalent neurological disorder that is characterized by recurrent unprovoked seizures, which are caused by instant abnormal hypersynchronous electrical activity in a
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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Fig. 6 – (A) Representative images of double immunofluorescence labeling for NG2 and the endogenous proliferation marker Ki67 in the hippocampi of rats in the control group (A1–A3) and the experimental group (B1–B3). The red arrows indicate NG2positive cells, the yellow arrows indicate Ki67-positive cells, and the white arrows indicate cells co-expressing both markers, representing dividing NG2-positive cells. As shown in the image, the NG2-positive cells in the experimental group exhibited a reactive status, which was characterized by enlarged cell bodies, numerous membrane blebs and many short filopodial-like processes. Scale bar: 100 lm. (B) The percentage of Ki67-positive OPCs was analyzed in the hippocampi of animals from the experimental groups and the control group. As in (B), the percentage of Ki67-positive OPCs began to increase immediately following SC in the acute phase, peaked during the latent phase, and then decreased during the chronic phase. Finally, the percentage of Ki67-positive OPCs in the experimental group was lower than that in the control group during the late chronic phase. This phenomenon indicates that the ability of OPCs to proliferate began to increase during the acute phase, peaked during the latent phase, and then decreased during the chronic phase.
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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neural network (Fisher et al., 2005). Previous studies of the underlying pathology of epilepsy have confirmed that myelin is affected in certain patients with epilepsy and in epileptic rats (Ye et al., 2013; You et al., 2011), indicating an association between epilepsy and myelin damage. The present study revealed that the myelin damage observed in the rat hippocampus began during the acute stage of lithium-pilocarpine-induced epileptogenesis and that the severity of this damage increased throughout the natural trajectory of epilepsy, leading to an abundance of demyelination. To further explore the mechanism resulting in an increase in myelin damage, changes in oligodendrocytes, which are myelin-forming cells, were detected. Our results showed that accompanying this myelin damage, the number of CC1-positive oligodendrocytes decreased throughout all phases of lithium-pilocarpine-induced epileptogenesis, indicating that the myelin damage is partly attributable to the ongoing death of CC1-positive oligodendrocytes. Because of the crucial role of myelin and oligodendrocytes in protecting and supporting the axon and because the integrity of myelin is crucial for maintaining normal neuronal network function and sequential electrical impulse conduction (Piaton et al., 2010), the loss of myelin and oligodendrocytes may contribute to the pathology of epileptogenesis. OPCs, which comprise approximately 5–8% of the total glial population in the adult CNS, are capable of generating myelinating oligodendrocytes during both brain development and adulthood (Levine et al., 2001; Polito and Reynolds, 2005). Although the function of OPCs is incompletely understood, one of their precise functions is to serve as a reservoir of new oligodendrocytes throughout life (Polito and Reynolds, 2005). In the normal adult brain, OPCs remain in a quiescent state in which these cells divide infrequently (Dawson et al., 2003). Upon myelin damage, such as that which occurs in experimental demyelination models, OPCs are rapidly recruited to the traumatized areas and ultimately differentiate into mature oligodendrocytes to restore myelin sheaths (Di Bello et al., 1999; Levine and Reynolds, 1999; Watanabe et al., 2002). The endogenous myelin repair process, termed remyelination, is primarily mediated by oligodendrocytes and OPCs. Based on immunofluorescence using NG2 and Ki67 antibodies, we also demonstrated that NG2-positive OPCs in the rat hippocampus underwent increased NG2 expression and morphological changes, such as enlargement of the soma and processes, which were observed during several phases (the acute, latent, and chronic phases) of epileptogenesis. Increased numbers of OPCs and an increased percentage of Ki67-positive OPCs were observed to be concurrent with these morphological changes, indicating that OPCs rapidly activated and proliferated after SC induced by lithium–pilocarpine. We can speculate on the possible implications of the changes observed in OPCs during the process of lithiumpilocarpine-induced epileptogenesis. Similar morphological changes in NG2-positive OPCs have been reported in some diseases that exhibit white matter injury, such as mechanical brain injury (Levine, 1994), focal ischemia (Tanaka et al., 2001), and experimental autoimmune encephalomyelitis (Nishiyama et al., 1997). These changes, including altered morphology, enhanced proliferation and increased NG2 protein expression, are generally regarded as indicators of the
activation of NG2-positive OPCs (Levine, 1994; Levine and Reynolds, 1999; Nishiyama et al., 1997). Because OPCs have the capacity to mature into myelinating oligodendrocytes for remyelination, the transition from a quiescent to an activated state may reflect an attempt to compensate for the loss of mature oligodendrocytes. Considering that this transition concurrently occurred with demyelination and oligodendrocyte loss during this experiment, we speculate that the activation of OPCs represent an attempt by OPCs to differentiate into mature oligodendrocytes to remyelinate damaged myelin, indicating that an endogenous repair process is initiated in the process of lithium-pilocarpine-induced epileptogenesis. However, as our results showed, the increased number of OPCS and their increased ability to proliferate were observed merely during several phases of epileptogenesis (the acute, latent, and chronic phases), indicating the activation of OPCs is just a transient process. In addition, despite the increased number of OPCs observed during several phases of epileptogenesis (the acute, latent, and chronic phases), myelin damage and the loss of oligodendrocytes were observed throughout epileptogenesis, indicating the lack of efficient endogenous repair. To explore the possible mechanism behind the transient activation of OPCs during epileptogenesis, we detected dynamic changes in two transcription factors, olig1 and olig2, which are crucial for the proliferation and differentiation of OPCs into mature oligodendrocytes during myelination and remyelination. Olig1 and olig2 are closely related bHLH transcription factors that are expressed in oligodendroglial lineage cells in the CNS (Lu et al., 2002). Knockout mouse studies have demonstrated that olig1 is required for the generation of mature oligodendrocytes and that olig2 plays a crucial role in the initial specification of the oligodendrocyte lineage during developmental myelination (Lu et al., 2002; Xin et al., 2005). Moreover, during remyelination in the adult brain, olig1 and olig2 continue to perform an important function. Arnett et al. found that the observed decrease in remyelination is caused by the impaired ability of OPCs to differentiate in olig1-/- mice after demyelination is induced by several toxins. This observation indicates that olig1 plays a crucial role in regulating oligodendrocyte differentiation and consequent remyelination in the adult brain (Arnett et al., 2004). Fancy et al. (2004) have demonstrated that the upregulation of the olig2 protein is critical to the activation of a quiescent population of OPCs following demyelination and enables these cells to mature into myelinating oligodendrocytes to repair damaged myelin sheaths. These findings indicate the crucial roles of olig1 and olig2 in enabling OPCs to differentiate into new oligodendrocytes to remyelinate damaged myelin sheaths. In the present study, we found that the expression levels of olig1 and olig2 were up-regulated during the acute phase, peaked during the latent phase, and then sharply declined during the chronic phase of epileptogenesis. Based on their functions in myelination and remyelination, we speculate that the increased expression of olig1 and olig2 during the early phases (the acute and latent phases) of epileptogenesis may reflect an attempt to assist activating OPCs to compensate for the loss of myelin that occurs during epileptogenesis. The sharp decline in olig1 and olig2 expression during the
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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chronic and late chronic phases, during which spontaneous recurrent seizures (SRS) occur, may contribute to the inhibition of OPC activation, proliferation and differentiation, resulting in the restriction of the endogenous repair of myelin during epileptogenesis. However, although our findings suggest that the activation of OPCs may contribute to the endogenous repair process by supplying new oligodendrocytes, we have no direct evidence that the activated OPCs are involved in remyelination. Further study is needed to address the following issues: (1) whether the activated OPCs successfully differentiate into mature oligodendrocytes and (2) the fate (death or differentiation) of the decreased population of OPCs in chronic and late chronic phases. Overall, our findings demonstrate for the first time that loss of myelin accumulates during lithium-pilocarpineinduced epileptogenesis. Myelin damage was accompanied by the loss of oligodendrocytes, indicating that myelin damage is partly secondary to the loss of oligodendrocytes. The activation of OPCs, although transient, indicates that an endogenous repair process is initiated during epileptogenesis and that the transient activation of OPCs may be partly due to changes in its related regulatory factors. Preventing damage to the myelin sheath and promoting the proliferation and differentiation of OPCs to participate in this endogenous repair process may provide a promising treatment strategy for epilepsy.
4.
Experimental procedures
4.1.
Animals
For this study, 106 adult Sprague–Dawley rats weighing 200– 250 g (Experimental Animal Center, Chongqing Medical University, Chongqing, China) were group housed and provided with ad libitum access to water and food. The animals were housed with a 12 h light/dark cycle and at a temperature of 22–24 1C and a relative humidity of 50–60%. The Administrative Panel on Laboratory Animal Care of Chongqing Medical University approved all experimental procedures.
4.2. Preparation of the epilepsy model and video–EEG monitoring The epilepsy modeling and video–EEG monitoring were conducted as previously described (Ye et al., 2013). One week before inducing seizure convulsion, the rats were anesthetized via intraperitoneal (i.p.) injection of 10% chloral hydrate (3 ml/kg) and placed in a stereotactic frame. To record cortical EEGs, a pair of insulated stainless steel electrodes was inserted into the skull bilaterally over the parietal cortex, and a ground lead was positioned over the nasal sinus. After four days of recovery, the rats were monitored via continuous cortical electroencephalography (EEG). All EEG recordings and video data were visually screened, and seizures were confirmed by trained human observers. For the preparation of the epilepsy animal model, SC was induced in the rats four days after surgery via i.p. injection of pilocarpine (pilo, 50 mg/kg, Sigma) 18 h after the injection of
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lithium (127 mg/kg, i.p, Solarbio, Beijing, China). To reduce peripheral effects, methyl-scopolamine was administered via an i.p. injection (1 mg/kg) 30 min prior to the pilo injection. Ninety minutes after SC, all rats received a single dose of diazepam (10 mg/kg, i.p.) The rats in the control group received an equivalent volume of 9% saline. To evaluate the dynamic changes in myelin and OPCs, the rats were sacrificed at various time points after SC reflecting the different phases of the natural trajectory of epilepsy: the acute phase of epileptogenesis was defined as the first 24 h after acute SC; the latent phase consisted of the following five days; the chronic phase was defined as the two weeks after the first spontaneous recurrent seizure (SRS); and the late chronic phase was defined as the two months after the first SRS (Mazzuferi et al., 2010). According to previous descriptions, the threshold discharge for the first spontaneous electrographic seizure was set at a frequency of 45 Hz, an amplitude of 42 baseline and a duration of 410 s (Goffin et al., 2007). The number of spontaneous seizures in the rats during the late chronic phase was monitored for one month after the first spontaneous seizure. Rats that did not exhibit SC, that died, or that did not develop spontaneous seizures were excluded.
4.3.
Immunofluorescence
The rats (8 animals per group) were anesthetized via an i.p. injection of 10% chloral hydrate (3 ml/kg) and were then transcardially perfused with 120 ml of 9% saline followed by 250 ml of 4% paraformaldehyde. After perfusion, each brain was removed and placed in 4% paraformaldehyde containing 30% sucrose at 4 1C until the brain sank. Sagittal sections were serially cut at a thickness of 40 μm using a freezing microtome and were stored in cryoprotectant solution at 4 1C until immunofluorescence staining was performed. The following primary antibodies/combinations of antibodies were used: NG2 (mouse monoclonal, Chemicon) and PDGFR-α (rabbit polyclonal, Santa Cruz Biotech), each at 1:200; CC1 (mouse monoclonal, Chemicon) at 1:200; Ki67 (rabbit polyclonal, Chemicon) and NG2 (same as above) at 1:1000 and 1:200, respectively; or NG2 alone (same as above) at 1:200. All of the antibodies listed were incubated with tissue overnight. All images were captured using a laser-scanning confocal microscope.
4.4.
Western blotting
The hippocampus of each rat (8 animals per group) was separately collected at the indicated phase of epileptogenesis (acute, latent, chronic, or late chronic phase) and stored in liquid nitrogen immediately after dissection. Total protein samples were extracted using a protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer's instructions, and the protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, USA). Equal amounts of protein (30 μg per lane) were loaded on 8–16% SDS polyacrylamide gels. After electrophoresis, the samples were transferred to polyvinylidene difluoride membranes (0.22 μm, Millipore Corp., Billerica, MA, USA) and the membranes were blocked for 1 h in PBS containing 10% skim
Please cite this article as: Luo, Y., et al., Alterations in hippocampal myelin and oligodendrocyte precursor cells during epileptogenesis. Brain Research (2015), http://dx.doi.org/10.1016/j.brainres.2015.09.027
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milk. The membranes were further incubated in primary antibodies overnight at 4 1C followed by incubation in peroxidase-conjugated secondary antibodies. The primary antibodies used were as follows: MBP (SMI99, Covance, Emeryville, CA, USA) at 1:2000; PDGFR-α (rabbit polyclonal, Santa Cruz Biotech) at 1:1000; olig1 (mouse monoclonal, Chemicon) at 1:1000; and olig2 (mouse monoclonal, Chemicon). After 35-min washes with PBS containing 0.1% Tween20 (PBS-T) on a shaker, the membranes were processed using enhanced chemiluminescence. To normalize the results, parallel Western blots probed with an anti-β-actin antibody (1:1000, Chemicon) were developed. The Western blot bands were analyzed via densitometry using ACDsee pro 2.5 and Image J software.
4.5.
Data collection and statistical analysis
EEG analysis was performed as previously described; additionally, discharges with frequencies of 45 Hz, amplitudes of 42 baseline, and durations of 410 s were regarded as spontaneous seizures (Goffin et al., 2007; Ye et al., 2013). For image analysis, the numbers of NG2-positive cells and CC1-positive cells were counted within uniform areas (200 microscopic fields) from various hippocampal regions (CA1, CA3, and DG). The numbers of NG2-positive cells, Ki67- and NG2- double positive cells and CC1-positive cells from at least five consecutive coronal sections in each animal (n¼ 8) were averaged. All data are presented as the means7SD and were analyzed via one-way ANOVA followed by either Dunnett's or Tukey's post hoc test to determine the statistical significance of the observed difference; po0.05 was considered statistically significant.
Author contributions Yuanyuan Luo and Li Jiang conceived and designed the experiments. Yuanyuan Luo performed the experiments and analyzed the data. Qiao Hu, Qian Zhang, Siqi Hong, Xiaoju Tang, Zhengxiong Yao, and Li Chen contributed reagents, materials, and analysis tools. Yuanyuan Luo wrote the manuscript.
Acknowledgments Q5 This work was supported by the National Natural Science
Foundation of China (NSFC Grant 81371452), the National Natural Science Foundation of Chongqing (CSCT 2013jjB0031), and the Research Fund for the Doctoral Program of Higher Education of China (20125503110011).
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