Cell Mol Neurobiol DOI 10.1007/s10571-015-0313-4
ORIGINAL RESEARCH
Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury Si Zhang1,2 • Peijun Ju3 • Editha Tjandra4 • Yeeshan Yeap5 • Hamed Owlanj4 Zhiwei Feng1
•
Received: 29 June 2015 / Accepted: 24 November 2015 Ó Springer Science+Business Media New York 2016
Abstract Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of
Si Zhang and Peijun Ju contributed equally to the paper.
Electronic supplementary material The online version of this article (doi:10.1007/s10571-015-0313-4) contains supplementary material, which is available to authorized users. & Zhiwei Feng [email protected]; [email protected] 1
School of Life Science and Technology, Xinxiang Medical University, Jinsui Road, Xinxiang 453003, Henan, China
2
Brain Research Center, Faculty of Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada
3
Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai 201108, China
4
School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
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Cytogenetics Laboratory, Singapore General Hospital, Singapore, Singapore
OX42 ? microglial cells and glial fibrillary acidic protein ? astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages. Keywords Spinal cord injury EGFR inhibition Remyelination
Introduction Spinal cord injury (SCI) was characterized by cell death and disruption of neuronal circuits, etc., which comprise primary and secondary phases (Profyris et al. 2004). Its prognosis is often poor due to incapacity of neuronal regeneration (Thuret et al. 2006; Tohda and Kuboyama 2011). However, recent studies revealed that inhibition of epidermal growth factor receptor (EGFR) pathway could promote axon regeneration in SCI and reduce reactive astrogliosis (Erschbamer et al. 2007; Koprivica et al. 2005; Li et al. 2011). As myelination plays a key role in functional integrity of axon and recovery of SCI, it would be worthwhile to investigate the effects of EGFR inhibition on improving myelination cascades in SCI (Berry et al. 2011; McDonald and Belegu 2006). The loss of oligodendrocytes (OLs) is a vital part of the cascading secondary events (Mekhail et al. 2012) and prominently contributes to axonal demyelination in SCI (McTigue and Tripathi 2008). In the recovery stage of SCI, the remyelination of the denuded axons was shown to be mainly achieved by the newly differentiated OLs from proliferative oligodendrocyte precursor cells (OPCs)
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(McTigue and Tripathi 2008; Nishiyama 1998). Thus, protection of the OPCs or stimulating their proliferation might favor myelination/remyelination in SCI (Franklin and Ffrench-Constant 2008; Rosenberg et al. 2005). The EGFR regulates a wide range of cellular activities (Merlino 1990) and were recently shown to be activated in SCI (Erschbamer et al. 2007). The role of EGFR was found to be involved in the activation of astrocytes and responsible for the expression of chondroitin sulfate proteoglycans (CSPGs) (Liu et al. 2006; Smith and Strunz 2005). Reactive astrocytes and CSPGs were thought as inhibitory elements for axon regeneration (Silver and Miller 2004). Thus, inhibition of EGFR pathways could reduce the reactive astrogliosis (Li et al. 2011) and promote the axon regeneration (Ju et al. 2012; Li et al. 2011). Besides, EGFR was also shown to reinforce the gliogenesis post injury of CNS (Aguirre et al. 2007; Liu and Neufeld 2004). Constitutive expression of EGFR is involved in accumulation of cells resemblance to OPCs (Ivkovic et al. 2008). Stimulated EGFR signaling had also been found to promote myelination (Aguirre et al. 2007). Such discrepancy in findings indicate the necessity for clarifying detailed roles of EGFR in a cell type-dependent manner in SCI. Interestingly, recent data has revealed that the therapeutic effects of EGFR inhibition mainly affected glial cells in SCI (Ahmed et al. 2009), though the treatment efficacy has been debated (Sharp et al. 2012). Here we demonstrated that EGFR inhibition promoted the growth and myelinating differentiation of OPCs. Meanwhile, the activation of astrocytes and microglial cells, as well as apoptosis of OPCs was reduced by EGFR inhibition.
laminectomy at T9 vertebral level. The T10 segment was then positioned using an adaptor to stabilize the spinal cord. Standard New York University weight-drop device was used for producing contusion injury by dropping a 10 g weight rod onto the exposed dura (Zhang et al. 2010). Drug Delivery 3 ll of PD168393 (1 mM) or PBS as vehicle control was injected into the center of injured region with a 5 ll Hamilton syringe immediately after contusion. To achieve the prolonged release of drug, a small block (3 mm 9 3 mm 9 3 mm) of gelatin matrix (Di Silvio et al. 1994) containing 7 ll PD168393 (10 mM) or PBS was put onto the damaged dorsal surface before suturing the muscles and skins (Solorio et al. 2010). After operation, animals were then placed on a heating pad until fully recovered from anesthesia, and housed singly per cage. The infections were prevented by daily administration of Ampicillin (1.5 mg/kg), starting immediately before surgery and lasting for 1 week. Carprofen (5 mg/kg) was also given daily via subcutaneous injection for 4 days postoperatively for pain management. The urinary bladders were manually expressed twice a day until the reliable reflex bladder emptying was successfully re-established. BrdU (5-Bromo-20 -Deoxyuridine) Administration Animals were given the thymidine analogue BrdU labeling reagent (1 ml/100 g, Invitrogen) twice at 2-h intervals on day 3 post injury, following the manufacturer’s instruction. The number of proliferative astrocytes and microglia were determined at 2 h post injection.
Materials and Methods Tissue Preparation Spinal Cord Injury Swiss Albino female mice of 7–8 weeks old from Laboratory Animal Center of National University of Singapore were used. They were housed in a specific antigen-free environment at 22 °C with a 12 h light/dark cycle and standard rodent diet ad libitum. All animal handlings and experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore. Mice were randomly assigned to three different groups: (1) sham-operated groups, (2) vehicle (PBS) control groups with SCI, and (3) the PD168393-treated groups with SCI. They were anesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/ kg). Once the animal was areflexic, the skin incision was made between T8 and T10 regions to expose the spine. The thoracic spinal cord was then exposed by performing a
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Mice were sacrificed at 3 days, 1, 2, or 3 weeks respectively after SCI with overdose of pentobarbital sodium, followed by transcardial perfusion with cold heparinized saline and then by 4 % paraformaldehyde (PF) in 0.1 M PBS (pH 7.4). Fixed spinal cords were dissected out and post-fixed in 4 % PF for a minimum of 2 days, followed by immersing in PBS with 30 % sucrose for cryoprotection. 6 mm spinal cords containing contusive epicenter were quickly embedded into Optimal Cutting Temperature compound (Sakura), and fast frozen in liquid nitrogen. Frozen sagittal sections (30 lm) were cut through the region encompassing the injury site with cryostat. Immunohistochemistry The sectioned samples were rinsed with PBS and blocked with 4 % bovine serum albumin (BSA) in 0.1 M PBS
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containing 0.1 % Triton X-100 (PBS/Tx) for 2 h. For immunohistochemistry experiment, sections were incubated with goat anti-MBP (myelin basic protein, Chemicon International) at 4 °C overnight. After rinsing with PBS, the sections were incubated with biotinylated donkey antigoat IgG (Vector Laboratories) for 2 h, and processed by Vectastain Elite ABC kit (Vector Laboratories), according to the manufacturer’s instruction. Brown coloring was developed with DAB (Sigma). After thorough rinsing, the sections were mounted onto slides, air-dried overnight and covered by PermountTM medium (EMS). To perform the Hematoxylin & Eosin staining, the sections were mounted onto slides coated with poly-Llysine and dried overnight. Mounted slides were incubated in 4 % PF and subsequently transferred into hematoxylin solution (Sigma-Aldrich), and rinsed in distilled water twice before being dipped in differentiating solution (1 % HCl in 100 % ethanol). The slides were then incubated in Scott’s tap water substitute (Magnesium sulfate buffered with sodium bicarbonate) (Electron Microscopy Sciences). The slides were then incubated in 95 % ethanol before being transferred to eosin Y solution (Sigma-Aldrich). They were placed in 70 % ethanol to remove excess eosin staining. After staining, slides were air-dried and covered with paramount (Innovex Biosciences), and viewed with bright-field microscope (Olympus). Immunofluorescence Staining For immunofluorescence staining, sections were permeabilized with PBS/Triton X-100 before being blocked with 4 % BSA in PBS/Tx, and were subsequently incubated overnight at 4 °C with the primary antibodies (Table S1) diluted in PBS/Tx/1 % BSA. The sections were washed with PBS and incubated with corresponding secondary antibodies. Finally, the sections were counterstained with DAPI, mounted and covered with FluorosaveÒ (Calbiochem) on slides. Staining for BrdU combined with different glial phenotypic markers was performed separately. In all the double-labeling procedures, the sections were firstly processed for marker staining (as stated above). Then the freefloating sections were treated with 1 N HCl to open the DNA structure. The sections were finally incubated in borate buffer (0.1 M, pH7.4), followed by BrdU staining with routine immunostaining protocol. For pEGFR staining, antigen retrieval was performed by incubating tissue sections in 0.3 % PBS-Tx and in 0.5 mM EDTA (pH 8.0) each, followed by routine immunostaining. Sampling, Cell Counting and Statistical Analysis All sections were imaged with confocal microscope (Carl Zeiss LSM510), and photomicrographs were taken by 910,
920 or 963 objective lenses. Five animals per group were examined at each time point. The sagittal sections were preferred in the medial region between midline and peripheral rim of the spinal cord. These sections covered most areas of grey matter, the dorsal and ventrolateral white matters, which include the regions for locomotor and sensory function. For individual quantification, at least three selected sections were examined for each slide. The cell counting was performed along the rostrocaudal axis from 3 mm rostral and -3 mm caudal of the injury epicenter in a blinded manner. Within the 6 mm range, continuous squares with 500 lm intervals were defined. Therefore, cells being measured for corresponding staining were from the specified area (0.25 mm2). The proportion of proliferative OX42 ? and glial fibrillary acidic protein (GFAP) ? cells was calculated in the -1.5 to 1.5 mm range of the injured region at 3 days post SCI. The OX42 ?/BrdU ? or GFAP ?/BrdU ? cells were counted as positive if the BrdU ? nucleus was unambiguously incorporated in nuclear region. To determine the level of MBP-immunoreactivity, computer-assisted image analysis was used to transform the color intensity of MBP immunoreactivity into values with Zen 2007 software (Carl Zeiss, Germany). All the sections were processed simultaneously with identical light beam and wavelength throughout the experiment. Statistical analysis of data was performed with a multiple-measurement ANOVA for comparison of different groups over time or a two-tailed Student’s t test. In all cases, the data were expressed as mean ± SD. A P value \ 0.05 was considered statistically significant.
Results EGFR Inhibition Promotes Remyelination in Mouse Spinal Cord Injury We found that local delivery of the EGFR inhibitor (PD168393, PD) into injured spinal segment could efficiently reduce the level of phosphorylated EGFR, especially in OX42 ? microglia and GFAP ? astrocytes (Fig. S1), and also improve the neural functional recovery of experimental contusive SCI (Ju et al. 2012). To further characterize the beneficial effects of PD in contusive SCI, we examined the histopathological changes of injured spinal segments with or without PD treatment. As shown in Fig. 1a-c, we found that the scar formation in PD-treated mice was much smaller than that in vehicle-treated ones at 3 weeks post injuries. These results suggest that EGFR inhibition can reduce tissue damage in SCI. Since myelination is important for functional integrity of axons, we then determined whether the myelin expression was
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Fig. 1 Effects of EGFR inhibition on gross histological change and myelin sparing post SCI. In control and PD168393 (PD)-treated group at 3 weeks post injury, histological changes were shown by HE staining (sagittal sections). The injured segment showed distinct cell loss, cell aggregation and scar formation at the injury epicenter (a–c). Immunostaining with anti-MBP antibody of spinal cords (coronal
sections) in both control (left panel) and PD-treated group (right panel) at 3 weeks post injury (d, e). MBP immunofluorescent staining of the injury site at 3, 7 and 14 days post SCI. Nuclei were counterstained with DAPI in all images (sagittal sections) (f–m). Quantitative analysis of MBP signals in the injured areas per mm2 shown in f–m (n). Scale bars 500 lm (a–e); 25 lm (f–m)
affected by PD. Immunohistochemical results showed that PD-treated mice had more MBP expression (a major constituent of the myelin sheath) than the vehicle-treated mice at 3 weeks post injury (Fig. 1d, e). Then detailed information of MBP expression was further evaluated, as shown in Fig. 1f–m. Though the injury caused remarkable myelin loss at early stage of SCI, a significant increase of MBP expression was observed in PD-treated mice from 1 week post injury (P \ 0.01, Fig. 1n). These findings suggest that EGFR inhibition could preserve myelination and/or promote remyelination.
The EGFR Inhibition Stimulates OPCs in SCI
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The growth of OLs is crucial for remyelination in SCI (Nishiyama 1998). To investigate if EGFR inhibition affects the development of OPCs, we labeled OPCs with NG2 proteoglycan and A2B5 which are conventional markers for OPCs. In agreement with results from other labs (Lytle and Wrathall 2007; McTigue et al. 2001), the number of NG2 ?/A2B5 ? OPCs increased in response to injury, and peaked at 7 days (Fig. 2a, c, e). However, upon EGFR inhibition, the NG2 ?/A2B5 ? OPCs count was
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substantially increased (Fig. 2b, d, f) and exhibited remarkable significance over 14 days of SCI (Fig. 2g, P \ 0.01). Consistently, the A2B5 ? cells also drastically increased with the onset of SCI, the number of which was significantly greater in PD-treated mice, as compared with vehicle-treated mice (Fig. 2h, P \ 0.01). Mature Oligodendrocytes was Highly Increased by EGFR Inhibition in SCI
the CC1 ? antibody were counted at 3 days, 1, 2, and 3 weeks post injuries (Fig. 3a–h). The density of CC1 ? cells decreased at 3 days and 1 week, but increased from 2 weeks (Fig. 3i). However, PD treatment effectively stimulated CC1 ? cells since 1 week post injury (1 week: P \ 0.01, 2 and 3 weeks: P \ 0.05), suggesting that EGFR inhibition can also increase the numbers of mature OLs in SCI. EGFR Inhibition Reduces Apoptosis of OPCs in SCI
The above effects of PD on the growth of OPCs and myelin sparing suggested that treatment with PD might increase the number of mature OLs. Thus, mature OLs labeled with
Fig. 2 A2B5 ?/NG2 ? OPCs at 3, 7 and 14 days of SCI. Immunostaining of A2B5 (red) and NG2 proteoglycan (green) in both control group (a, c, e) and PD-treated group (b, d, f) at 3, 7 and 14 days post SCI. Nuclei were counterstained with DAPI in all images. The number of NG2 ?/A2B5 ? cells was quantitatively compared
To investigate if the higher number of OPCs was also attributed to protection of OPCs in PD-treated mice, we
between control and PD-treated groups in 3, 7 and 14 days post injury (g). Quantitative analysis of A2B5 immunopositive cells between control and PD-treated groups in 3, 7 and 14 days post injury (h). Scale bars 50 lm
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Fig. 3 The CC1 ? oligodendrocytes are increased by PD treatment. Immunostaining of CC1 (red) in both control group (a, c, e, g) and PD-treated group (b, d, f, h) at 3, 7, 14 and 21 days post SCI. Nuclei
were counterstained with DAPI in all images. Quantification of CC1 ? cells in above experiments (i). Scale bars 50 lm
examined the apoptosis of OPCs in SCI. We examined the co-localization of activated caspase-3 (apoptotic marker) with OPC marker (A2B5) at 3 and 7 days post injury, when the major apoptotic cell death occurred (Liu et al. 1997). As shown in Fig. 4a–e, more apoptotic A2B5 cells was found in vehicle group (P \ 0.01). Quantitative analysis further revealed that the percentage of apoptotic OPCs in proportion to total OPCs in PD-treated mice decreased to 12.1 % from 35.8 % in vehicle treated mice at 3 days post injury (Fig. 4f, P \ 0.01). Therefore, EGFR inhibition can protect OPCs from apoptosis in SCI, suggesting that the growth of OPCs by EGFR inhibition may benefit from both cell protection and cell growth.
EGFR Inhibition Reduces the Proliferation of OX42 1 Microglia and GFAP 1 Astroyctes
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As the hypertrophy of microglia and astrocytes is the major factor causing secondary damage of SCI, we examined the proliferation of OX42 ? microglia or GFAP ? astrocytes by BrdU labeling. At 3 days post SCI, the numbers of proliferating microglia and astrocytes were drastically increased. However, the proliferating OX42 ? or GFAP ? cells were less in PD-treated animals (Fig. 5e, P \ 0.05 and Fig. 5f, P \ 0.01). Representative data of double immunofluorescent staining revealed less BrdU ? OX42 ? and GFAP ?/BrdU ? cells in PD-
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Fig. 4 PD alleviates OPC apoptosis in SCI. Z-stack confocal images of tissue harvested from control and PD-treated mice at 3 (a, c) and 7 days (b, d) post injury was stained with antibodies against caspase3 (green) and A2B5 (red). Nuclei were counterstained with DAPI in all images. Quantification of apoptotic A2B5 ? oligodendrocyte progenitors within the 6 mm along rostral-caudal axis of spinal cord (e) (n = 5, **P \ 0.01). The proportion of caspase3 ?/A2B5 ? cells
in A2B5 ? cells per mm2 was determined by dividing the number of cells double-labeled for caspase-3 and A2B5 by the total number of A2B5 ? cells in control and PD-treated group respectively (f) (n = 5, **P \ 0.01). Images showed z-axis projections of 23 9 0.40 lm (a), 22 9 0.55 lm (b), 25 9 0.40 lm (c), 21 9 0.40 lm (d). Scale bars 10 lm
treated mice at 3 days (Fig. 5a–d). These results indicate the reduction of proliferative microglia/macrophages and astrocytes by EGFR inhibition in SCI. The OX42 ? cells post SCI was further evaluated by examining the OX42 immunoflorescent staining in the lesion site of spinal cord epicenter. As shown in Fig. 6b, d, there was remarkable decrease of OX42 immunoreactivity in PD-treated mice at both 3 and 21 days post injury compared with those in
vehicle-treated mice (Fig. 6a, c). Quantitative results showed that the reduction of OX42 ? cells was significant throughout the whole lesion region in PD-treated mice in comparison with the vehicle-treated mice at both 3 and 21 days post injury (Fig. 6e, f, P \ 0.01, multiple measurement ANOVA). Therefore, PD168393 treatment could attenuate the reaction of OX42 ? microglia/macrophages after SCI.
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Fig. 5 EGFR inhibition reduces the proliferative OX42 ? macrophages and GFAP ? astrocytes at 3 days post injury. Immunofluorescent images showed the BrdU ?/OX42 ? (a, b), BrdU/ GFAP ? (c, d) cells in the injury epicenter in both control and PDtreated groups. Nuclei were counterstained with DAPI. Examples of
double-labeled OX42 ?/BrdU ? cells and GFAP ?/BrdU ? cells were indicated by arrow. Examples of BrdU? only cells were indicated by arrowhead. The results were confirmed by quantitative analysis of number counting in (e) and (f) (n = 5, *P \ 0.05, **P \ 0.01). Scale bars 50 lm
Discussion
Despite that manipulating myelination has been thought as an effective strategy for the treatment of SCI (Wu and Ren 2008), few successful approaches are proposed so far. It has been well demonstrated that the loss of OLs in SCI would lead to demyelination; on the other hand, remyelination is attributed to the recruitment of OPCs after injury (Grossman et al. 2001; Levine et al. 2001). In our study, we detected more OLs and OPCs at different time-point in the PD-treated mice. Interestingly, high level of MBP was found expressed by the increasing number of OPCs in the PD-treated mice (Fig. S2). It has been reported that the differentiation of OPCs in SCI is the major cellular source for remyelination (Levine et al. 2001). These results suggested that inhibition of EGFR could stimulate the growth of OPCs which in turn participates the remyelination on axons in the injured spinal segment. On the other hand, we detected high level of MBP at very early stage of SCI, when the growth of OPCs was slow. We also found that the total density of CC1 ? OLs was higher in PD-treated mice at 3 days post injury. Moreover, there was a significantly reduced apoptosis of OLs and OPCs by EGFR inhibition
EGFR has been demonstrated to play an important role in SCI, such as activating astrocytes (Liu and Neufeld 2004), regulating the migration, proliferation and differentiation of glial progenitors (Aguirre et al. 2007; Aguirre and Gallo 2007), as well as enhancing the inflammatory responses of microglia (Li et al. 2011). Inhibition of EGFR, however, was recently shown to promote regeneration of injured axons (Koprivica et al. 2005). In the present study, we provided evidence for the first time that EGFR inhibition may remarkably increase the myelination in SCI. With the acute and local EGFR inhibition, high level of MBP expression was found in the injured segment since 3 days post SCI and kept increasing afterwards. Axon myelination is one of the important factors maintaining structural and functional integrity of neural circuits in spinal cords (Waxman 1989). The increased myelination by EGFR inhibition may significantly contribute to neural functional recovery as demonstrated in previous studies (Li et al. 2011; Nishiyama 1998).
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Fig. 6 PD treatment down-regulates the number of OX42 ? cells. The panoramic images of sections at 3 and 21 days post SCI were shown in control (a, c) and PD-treated (b, d) group. Quantification
OX42 ? cells in control (black columns) and PD-treated injured segment (white columns) at 3 or 21 days of injury were shown in (e) and (f). Scale bars 500 lm
(Fig. 4 and Fig. S3). These data collectively suggest that PD may also protect cells involved in remyelination. Therefore, the remyelination effect of the EGFR inhibition may include reservation of myelination during early stage and growth of OPCs for remyelination during chronic stage of SCI. The spontaneous remyelination in SCI is rare and context-dependent. It often becomes incomplete and fails even though OPCs are present on site (Trapp and Nave 2008). Due to the progressive gliosis in the demyelinated areas in SCI, an almost-immediate increase and activation of microglia/macrophages and astrocytes could be found therein (Zai and Wrathall 2005). They could release a multitude of cytokines, including the proinflammatory cytokines (Bezzi and Volterra 2001; Pineau and Lacroix 2007; Wang and Bordey 2008), which further activate the death signaling cascades in spinal cord (Kigerl et al. 2009; Shuman et al. 1997). Therefore, blocking the secondary damage in the injury niche might protect the OLs/OPCs from apoptosis. In this study, we confirm the inhibitory effect of PD on the activation of astrocytes and microglia/macrophages. Those effects may contribute to the glial
cell survival, and enhance remyelination together with axon regeneration in turn. In this cellular and molecular context, EGFR inhibitor PD emerged as a cytoprotective agent on OLs lineage owing in part to the inhibition of the secondary damage from astrocytes and microglia. In summary, inhibition of EGFR signaling pathway can reduce tissue damage at both acute and subacute stages of SCI. The therapeutic mechanisms include ameliorating the secondary injury from microglia and astrocytes activation together with regulating growth and differentiation of OLs lineage. Application of EGFR inhibitor after SCI could be a promising approach to promote remyelination, axonal regeneration and functional recovery.
Funding The study was funded by National Natural Science Foundation of China (No. 81470053) and Natural Science Foundation of Shanghai, China (No. 14ZR1435900). Compliance with Ethical Standards Conflict of interest All authors declare that there are no conflicts of interest.
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Cell Mol Neurobiol Ethical approval All procedures performed in the study involving animals were in accordance with the ethical standards of Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore and Xinxiang Medical University, China.
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ORIGINAL RESEARCH
Inhibition of Epidermal Growth Factor Receptor Improves Myelination and Attenuates Tissue Damage of Spinal Cord Injury Si Zhang1,2 • Peijun Ju3 • Editha Tjandra4 • Yeeshan Yeap5 • Hamed Owlanj4 Zhiwei Feng1
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Received: 29 June 2015 / Accepted: 24 November 2015 Ó Springer Science+Business Media New York 2016
Abstract Preventing demyelination and promoting remyelination of denuded axons are promising therapeutic strategies for spinal cord injury (SCI). Epidermal growth factor receptor (EGFR) inhibition was reported to benefit the neural functional recovery and the axon regeneration after SCI. However, its role in de- and remyelination of axons in injured spinal cord is unclear. In the present study, we evaluated the effects of EGFR inhibitor, PD168393 (PD), on the myelination in mouse contusive SCI model. We found that expression of myelin basic protein (MBP) in the injured spinal cords of PD treated mice was remarkably elevated. The density of glial precursor cells and oligodendrocytes (OLs) was increased and the cell apoptosis in lesions was attenuated after PD168393 treatment. Moreover, PD168393 treatment reduced both the numbers of
Si Zhang and Peijun Ju contributed equally to the paper.
Electronic supplementary material The online version of this article (doi:10.1007/s10571-015-0313-4) contains supplementary material, which is available to authorized users. & Zhiwei Feng [email protected]; [email protected] 1
School of Life Science and Technology, Xinxiang Medical University, Jinsui Road, Xinxiang 453003, Henan, China
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Brain Research Center, Faculty of Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada
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Shanghai Key Laboratory of Psychotic Disorders, Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, Shanghai 201108, China
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School of Biological Sciences, Nanyang Technological University, Singapore, Singapore
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Cytogenetics Laboratory, Singapore General Hospital, Singapore, Singapore
OX42 ? microglial cells and glial fibrillary acidic protein ? astrocytes in damaged area of spinal cords. We thus conclude that the therapeutic effects of EGFR inhibition after SCI involves facilitating remyelination of the injured spinal cord, increasing of oligodendrocyte precursor cells and OLs, as well as suppressing the activation of astrocytes and microglia/macrophages. Keywords Spinal cord injury EGFR inhibition Remyelination
Introduction Spinal cord injury (SCI) was characterized by cell death and disruption of neuronal circuits, etc., which comprise primary and secondary phases (Profyris et al. 2004). Its prognosis is often poor due to incapacity of neuronal regeneration (Thuret et al. 2006; Tohda and Kuboyama 2011). However, recent studies revealed that inhibition of epidermal growth factor receptor (EGFR) pathway could promote axon regeneration in SCI and reduce reactive astrogliosis (Erschbamer et al. 2007; Koprivica et al. 2005; Li et al. 2011). As myelination plays a key role in functional integrity of axon and recovery of SCI, it would be worthwhile to investigate the effects of EGFR inhibition on improving myelination cascades in SCI (Berry et al. 2011; McDonald and Belegu 2006). The loss of oligodendrocytes (OLs) is a vital part of the cascading secondary events (Mekhail et al. 2012) and prominently contributes to axonal demyelination in SCI (McTigue and Tripathi 2008). In the recovery stage of SCI, the remyelination of the denuded axons was shown to be mainly achieved by the newly differentiated OLs from proliferative oligodendrocyte precursor cells (OPCs)
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(McTigue and Tripathi 2008; Nishiyama 1998). Thus, protection of the OPCs or stimulating their proliferation might favor myelination/remyelination in SCI (Franklin and Ffrench-Constant 2008; Rosenberg et al. 2005). The EGFR regulates a wide range of cellular activities (Merlino 1990) and were recently shown to be activated in SCI (Erschbamer et al. 2007). The role of EGFR was found to be involved in the activation of astrocytes and responsible for the expression of chondroitin sulfate proteoglycans (CSPGs) (Liu et al. 2006; Smith and Strunz 2005). Reactive astrocytes and CSPGs were thought as inhibitory elements for axon regeneration (Silver and Miller 2004). Thus, inhibition of EGFR pathways could reduce the reactive astrogliosis (Li et al. 2011) and promote the axon regeneration (Ju et al. 2012; Li et al. 2011). Besides, EGFR was also shown to reinforce the gliogenesis post injury of CNS (Aguirre et al. 2007; Liu and Neufeld 2004). Constitutive expression of EGFR is involved in accumulation of cells resemblance to OPCs (Ivkovic et al. 2008). Stimulated EGFR signaling had also been found to promote myelination (Aguirre et al. 2007). Such discrepancy in findings indicate the necessity for clarifying detailed roles of EGFR in a cell type-dependent manner in SCI. Interestingly, recent data has revealed that the therapeutic effects of EGFR inhibition mainly affected glial cells in SCI (Ahmed et al. 2009), though the treatment efficacy has been debated (Sharp et al. 2012). Here we demonstrated that EGFR inhibition promoted the growth and myelinating differentiation of OPCs. Meanwhile, the activation of astrocytes and microglial cells, as well as apoptosis of OPCs was reduced by EGFR inhibition.
laminectomy at T9 vertebral level. The T10 segment was then positioned using an adaptor to stabilize the spinal cord. Standard New York University weight-drop device was used for producing contusion injury by dropping a 10 g weight rod onto the exposed dura (Zhang et al. 2010). Drug Delivery 3 ll of PD168393 (1 mM) or PBS as vehicle control was injected into the center of injured region with a 5 ll Hamilton syringe immediately after contusion. To achieve the prolonged release of drug, a small block (3 mm 9 3 mm 9 3 mm) of gelatin matrix (Di Silvio et al. 1994) containing 7 ll PD168393 (10 mM) or PBS was put onto the damaged dorsal surface before suturing the muscles and skins (Solorio et al. 2010). After operation, animals were then placed on a heating pad until fully recovered from anesthesia, and housed singly per cage. The infections were prevented by daily administration of Ampicillin (1.5 mg/kg), starting immediately before surgery and lasting for 1 week. Carprofen (5 mg/kg) was also given daily via subcutaneous injection for 4 days postoperatively for pain management. The urinary bladders were manually expressed twice a day until the reliable reflex bladder emptying was successfully re-established. BrdU (5-Bromo-20 -Deoxyuridine) Administration Animals were given the thymidine analogue BrdU labeling reagent (1 ml/100 g, Invitrogen) twice at 2-h intervals on day 3 post injury, following the manufacturer’s instruction. The number of proliferative astrocytes and microglia were determined at 2 h post injection.
Materials and Methods Tissue Preparation Spinal Cord Injury Swiss Albino female mice of 7–8 weeks old from Laboratory Animal Center of National University of Singapore were used. They were housed in a specific antigen-free environment at 22 °C with a 12 h light/dark cycle and standard rodent diet ad libitum. All animal handlings and experimental procedures were approved by the Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore. Mice were randomly assigned to three different groups: (1) sham-operated groups, (2) vehicle (PBS) control groups with SCI, and (3) the PD168393-treated groups with SCI. They were anesthetized with an intraperitoneal (i.p.) injection of ketamine (100 mg/kg) and xylazine (10 mg/ kg). Once the animal was areflexic, the skin incision was made between T8 and T10 regions to expose the spine. The thoracic spinal cord was then exposed by performing a
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Mice were sacrificed at 3 days, 1, 2, or 3 weeks respectively after SCI with overdose of pentobarbital sodium, followed by transcardial perfusion with cold heparinized saline and then by 4 % paraformaldehyde (PF) in 0.1 M PBS (pH 7.4). Fixed spinal cords were dissected out and post-fixed in 4 % PF for a minimum of 2 days, followed by immersing in PBS with 30 % sucrose for cryoprotection. 6 mm spinal cords containing contusive epicenter were quickly embedded into Optimal Cutting Temperature compound (Sakura), and fast frozen in liquid nitrogen. Frozen sagittal sections (30 lm) were cut through the region encompassing the injury site with cryostat. Immunohistochemistry The sectioned samples were rinsed with PBS and blocked with 4 % bovine serum albumin (BSA) in 0.1 M PBS
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containing 0.1 % Triton X-100 (PBS/Tx) for 2 h. For immunohistochemistry experiment, sections were incubated with goat anti-MBP (myelin basic protein, Chemicon International) at 4 °C overnight. After rinsing with PBS, the sections were incubated with biotinylated donkey antigoat IgG (Vector Laboratories) for 2 h, and processed by Vectastain Elite ABC kit (Vector Laboratories), according to the manufacturer’s instruction. Brown coloring was developed with DAB (Sigma). After thorough rinsing, the sections were mounted onto slides, air-dried overnight and covered by PermountTM medium (EMS). To perform the Hematoxylin & Eosin staining, the sections were mounted onto slides coated with poly-Llysine and dried overnight. Mounted slides were incubated in 4 % PF and subsequently transferred into hematoxylin solution (Sigma-Aldrich), and rinsed in distilled water twice before being dipped in differentiating solution (1 % HCl in 100 % ethanol). The slides were then incubated in Scott’s tap water substitute (Magnesium sulfate buffered with sodium bicarbonate) (Electron Microscopy Sciences). The slides were then incubated in 95 % ethanol before being transferred to eosin Y solution (Sigma-Aldrich). They were placed in 70 % ethanol to remove excess eosin staining. After staining, slides were air-dried and covered with paramount (Innovex Biosciences), and viewed with bright-field microscope (Olympus). Immunofluorescence Staining For immunofluorescence staining, sections were permeabilized with PBS/Triton X-100 before being blocked with 4 % BSA in PBS/Tx, and were subsequently incubated overnight at 4 °C with the primary antibodies (Table S1) diluted in PBS/Tx/1 % BSA. The sections were washed with PBS and incubated with corresponding secondary antibodies. Finally, the sections were counterstained with DAPI, mounted and covered with FluorosaveÒ (Calbiochem) on slides. Staining for BrdU combined with different glial phenotypic markers was performed separately. In all the double-labeling procedures, the sections were firstly processed for marker staining (as stated above). Then the freefloating sections were treated with 1 N HCl to open the DNA structure. The sections were finally incubated in borate buffer (0.1 M, pH7.4), followed by BrdU staining with routine immunostaining protocol. For pEGFR staining, antigen retrieval was performed by incubating tissue sections in 0.3 % PBS-Tx and in 0.5 mM EDTA (pH 8.0) each, followed by routine immunostaining. Sampling, Cell Counting and Statistical Analysis All sections were imaged with confocal microscope (Carl Zeiss LSM510), and photomicrographs were taken by 910,
920 or 963 objective lenses. Five animals per group were examined at each time point. The sagittal sections were preferred in the medial region between midline and peripheral rim of the spinal cord. These sections covered most areas of grey matter, the dorsal and ventrolateral white matters, which include the regions for locomotor and sensory function. For individual quantification, at least three selected sections were examined for each slide. The cell counting was performed along the rostrocaudal axis from 3 mm rostral and -3 mm caudal of the injury epicenter in a blinded manner. Within the 6 mm range, continuous squares with 500 lm intervals were defined. Therefore, cells being measured for corresponding staining were from the specified area (0.25 mm2). The proportion of proliferative OX42 ? and glial fibrillary acidic protein (GFAP) ? cells was calculated in the -1.5 to 1.5 mm range of the injured region at 3 days post SCI. The OX42 ?/BrdU ? or GFAP ?/BrdU ? cells were counted as positive if the BrdU ? nucleus was unambiguously incorporated in nuclear region. To determine the level of MBP-immunoreactivity, computer-assisted image analysis was used to transform the color intensity of MBP immunoreactivity into values with Zen 2007 software (Carl Zeiss, Germany). All the sections were processed simultaneously with identical light beam and wavelength throughout the experiment. Statistical analysis of data was performed with a multiple-measurement ANOVA for comparison of different groups over time or a two-tailed Student’s t test. In all cases, the data were expressed as mean ± SD. A P value \ 0.05 was considered statistically significant.
Results EGFR Inhibition Promotes Remyelination in Mouse Spinal Cord Injury We found that local delivery of the EGFR inhibitor (PD168393, PD) into injured spinal segment could efficiently reduce the level of phosphorylated EGFR, especially in OX42 ? microglia and GFAP ? astrocytes (Fig. S1), and also improve the neural functional recovery of experimental contusive SCI (Ju et al. 2012). To further characterize the beneficial effects of PD in contusive SCI, we examined the histopathological changes of injured spinal segments with or without PD treatment. As shown in Fig. 1a-c, we found that the scar formation in PD-treated mice was much smaller than that in vehicle-treated ones at 3 weeks post injuries. These results suggest that EGFR inhibition can reduce tissue damage in SCI. Since myelination is important for functional integrity of axons, we then determined whether the myelin expression was
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Fig. 1 Effects of EGFR inhibition on gross histological change and myelin sparing post SCI. In control and PD168393 (PD)-treated group at 3 weeks post injury, histological changes were shown by HE staining (sagittal sections). The injured segment showed distinct cell loss, cell aggregation and scar formation at the injury epicenter (a–c). Immunostaining with anti-MBP antibody of spinal cords (coronal
sections) in both control (left panel) and PD-treated group (right panel) at 3 weeks post injury (d, e). MBP immunofluorescent staining of the injury site at 3, 7 and 14 days post SCI. Nuclei were counterstained with DAPI in all images (sagittal sections) (f–m). Quantitative analysis of MBP signals in the injured areas per mm2 shown in f–m (n). Scale bars 500 lm (a–e); 25 lm (f–m)
affected by PD. Immunohistochemical results showed that PD-treated mice had more MBP expression (a major constituent of the myelin sheath) than the vehicle-treated mice at 3 weeks post injury (Fig. 1d, e). Then detailed information of MBP expression was further evaluated, as shown in Fig. 1f–m. Though the injury caused remarkable myelin loss at early stage of SCI, a significant increase of MBP expression was observed in PD-treated mice from 1 week post injury (P \ 0.01, Fig. 1n). These findings suggest that EGFR inhibition could preserve myelination and/or promote remyelination.
The EGFR Inhibition Stimulates OPCs in SCI
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The growth of OLs is crucial for remyelination in SCI (Nishiyama 1998). To investigate if EGFR inhibition affects the development of OPCs, we labeled OPCs with NG2 proteoglycan and A2B5 which are conventional markers for OPCs. In agreement with results from other labs (Lytle and Wrathall 2007; McTigue et al. 2001), the number of NG2 ?/A2B5 ? OPCs increased in response to injury, and peaked at 7 days (Fig. 2a, c, e). However, upon EGFR inhibition, the NG2 ?/A2B5 ? OPCs count was
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substantially increased (Fig. 2b, d, f) and exhibited remarkable significance over 14 days of SCI (Fig. 2g, P \ 0.01). Consistently, the A2B5 ? cells also drastically increased with the onset of SCI, the number of which was significantly greater in PD-treated mice, as compared with vehicle-treated mice (Fig. 2h, P \ 0.01). Mature Oligodendrocytes was Highly Increased by EGFR Inhibition in SCI
the CC1 ? antibody were counted at 3 days, 1, 2, and 3 weeks post injuries (Fig. 3a–h). The density of CC1 ? cells decreased at 3 days and 1 week, but increased from 2 weeks (Fig. 3i). However, PD treatment effectively stimulated CC1 ? cells since 1 week post injury (1 week: P \ 0.01, 2 and 3 weeks: P \ 0.05), suggesting that EGFR inhibition can also increase the numbers of mature OLs in SCI. EGFR Inhibition Reduces Apoptosis of OPCs in SCI
The above effects of PD on the growth of OPCs and myelin sparing suggested that treatment with PD might increase the number of mature OLs. Thus, mature OLs labeled with
Fig. 2 A2B5 ?/NG2 ? OPCs at 3, 7 and 14 days of SCI. Immunostaining of A2B5 (red) and NG2 proteoglycan (green) in both control group (a, c, e) and PD-treated group (b, d, f) at 3, 7 and 14 days post SCI. Nuclei were counterstained with DAPI in all images. The number of NG2 ?/A2B5 ? cells was quantitatively compared
To investigate if the higher number of OPCs was also attributed to protection of OPCs in PD-treated mice, we
between control and PD-treated groups in 3, 7 and 14 days post injury (g). Quantitative analysis of A2B5 immunopositive cells between control and PD-treated groups in 3, 7 and 14 days post injury (h). Scale bars 50 lm
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Fig. 3 The CC1 ? oligodendrocytes are increased by PD treatment. Immunostaining of CC1 (red) in both control group (a, c, e, g) and PD-treated group (b, d, f, h) at 3, 7, 14 and 21 days post SCI. Nuclei
were counterstained with DAPI in all images. Quantification of CC1 ? cells in above experiments (i). Scale bars 50 lm
examined the apoptosis of OPCs in SCI. We examined the co-localization of activated caspase-3 (apoptotic marker) with OPC marker (A2B5) at 3 and 7 days post injury, when the major apoptotic cell death occurred (Liu et al. 1997). As shown in Fig. 4a–e, more apoptotic A2B5 cells was found in vehicle group (P \ 0.01). Quantitative analysis further revealed that the percentage of apoptotic OPCs in proportion to total OPCs in PD-treated mice decreased to 12.1 % from 35.8 % in vehicle treated mice at 3 days post injury (Fig. 4f, P \ 0.01). Therefore, EGFR inhibition can protect OPCs from apoptosis in SCI, suggesting that the growth of OPCs by EGFR inhibition may benefit from both cell protection and cell growth.
EGFR Inhibition Reduces the Proliferation of OX42 1 Microglia and GFAP 1 Astroyctes
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As the hypertrophy of microglia and astrocytes is the major factor causing secondary damage of SCI, we examined the proliferation of OX42 ? microglia or GFAP ? astrocytes by BrdU labeling. At 3 days post SCI, the numbers of proliferating microglia and astrocytes were drastically increased. However, the proliferating OX42 ? or GFAP ? cells were less in PD-treated animals (Fig. 5e, P \ 0.05 and Fig. 5f, P \ 0.01). Representative data of double immunofluorescent staining revealed less BrdU ? OX42 ? and GFAP ?/BrdU ? cells in PD-
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Fig. 4 PD alleviates OPC apoptosis in SCI. Z-stack confocal images of tissue harvested from control and PD-treated mice at 3 (a, c) and 7 days (b, d) post injury was stained with antibodies against caspase3 (green) and A2B5 (red). Nuclei were counterstained with DAPI in all images. Quantification of apoptotic A2B5 ? oligodendrocyte progenitors within the 6 mm along rostral-caudal axis of spinal cord (e) (n = 5, **P \ 0.01). The proportion of caspase3 ?/A2B5 ? cells
in A2B5 ? cells per mm2 was determined by dividing the number of cells double-labeled for caspase-3 and A2B5 by the total number of A2B5 ? cells in control and PD-treated group respectively (f) (n = 5, **P \ 0.01). Images showed z-axis projections of 23 9 0.40 lm (a), 22 9 0.55 lm (b), 25 9 0.40 lm (c), 21 9 0.40 lm (d). Scale bars 10 lm
treated mice at 3 days (Fig. 5a–d). These results indicate the reduction of proliferative microglia/macrophages and astrocytes by EGFR inhibition in SCI. The OX42 ? cells post SCI was further evaluated by examining the OX42 immunoflorescent staining in the lesion site of spinal cord epicenter. As shown in Fig. 6b, d, there was remarkable decrease of OX42 immunoreactivity in PD-treated mice at both 3 and 21 days post injury compared with those in
vehicle-treated mice (Fig. 6a, c). Quantitative results showed that the reduction of OX42 ? cells was significant throughout the whole lesion region in PD-treated mice in comparison with the vehicle-treated mice at both 3 and 21 days post injury (Fig. 6e, f, P \ 0.01, multiple measurement ANOVA). Therefore, PD168393 treatment could attenuate the reaction of OX42 ? microglia/macrophages after SCI.
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Fig. 5 EGFR inhibition reduces the proliferative OX42 ? macrophages and GFAP ? astrocytes at 3 days post injury. Immunofluorescent images showed the BrdU ?/OX42 ? (a, b), BrdU/ GFAP ? (c, d) cells in the injury epicenter in both control and PDtreated groups. Nuclei were counterstained with DAPI. Examples of
double-labeled OX42 ?/BrdU ? cells and GFAP ?/BrdU ? cells were indicated by arrow. Examples of BrdU? only cells were indicated by arrowhead. The results were confirmed by quantitative analysis of number counting in (e) and (f) (n = 5, *P \ 0.05, **P \ 0.01). Scale bars 50 lm
Discussion
Despite that manipulating myelination has been thought as an effective strategy for the treatment of SCI (Wu and Ren 2008), few successful approaches are proposed so far. It has been well demonstrated that the loss of OLs in SCI would lead to demyelination; on the other hand, remyelination is attributed to the recruitment of OPCs after injury (Grossman et al. 2001; Levine et al. 2001). In our study, we detected more OLs and OPCs at different time-point in the PD-treated mice. Interestingly, high level of MBP was found expressed by the increasing number of OPCs in the PD-treated mice (Fig. S2). It has been reported that the differentiation of OPCs in SCI is the major cellular source for remyelination (Levine et al. 2001). These results suggested that inhibition of EGFR could stimulate the growth of OPCs which in turn participates the remyelination on axons in the injured spinal segment. On the other hand, we detected high level of MBP at very early stage of SCI, when the growth of OPCs was slow. We also found that the total density of CC1 ? OLs was higher in PD-treated mice at 3 days post injury. Moreover, there was a significantly reduced apoptosis of OLs and OPCs by EGFR inhibition
EGFR has been demonstrated to play an important role in SCI, such as activating astrocytes (Liu and Neufeld 2004), regulating the migration, proliferation and differentiation of glial progenitors (Aguirre et al. 2007; Aguirre and Gallo 2007), as well as enhancing the inflammatory responses of microglia (Li et al. 2011). Inhibition of EGFR, however, was recently shown to promote regeneration of injured axons (Koprivica et al. 2005). In the present study, we provided evidence for the first time that EGFR inhibition may remarkably increase the myelination in SCI. With the acute and local EGFR inhibition, high level of MBP expression was found in the injured segment since 3 days post SCI and kept increasing afterwards. Axon myelination is one of the important factors maintaining structural and functional integrity of neural circuits in spinal cords (Waxman 1989). The increased myelination by EGFR inhibition may significantly contribute to neural functional recovery as demonstrated in previous studies (Li et al. 2011; Nishiyama 1998).
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Fig. 6 PD treatment down-regulates the number of OX42 ? cells. The panoramic images of sections at 3 and 21 days post SCI were shown in control (a, c) and PD-treated (b, d) group. Quantification
OX42 ? cells in control (black columns) and PD-treated injured segment (white columns) at 3 or 21 days of injury were shown in (e) and (f). Scale bars 500 lm
(Fig. 4 and Fig. S3). These data collectively suggest that PD may also protect cells involved in remyelination. Therefore, the remyelination effect of the EGFR inhibition may include reservation of myelination during early stage and growth of OPCs for remyelination during chronic stage of SCI. The spontaneous remyelination in SCI is rare and context-dependent. It often becomes incomplete and fails even though OPCs are present on site (Trapp and Nave 2008). Due to the progressive gliosis in the demyelinated areas in SCI, an almost-immediate increase and activation of microglia/macrophages and astrocytes could be found therein (Zai and Wrathall 2005). They could release a multitude of cytokines, including the proinflammatory cytokines (Bezzi and Volterra 2001; Pineau and Lacroix 2007; Wang and Bordey 2008), which further activate the death signaling cascades in spinal cord (Kigerl et al. 2009; Shuman et al. 1997). Therefore, blocking the secondary damage in the injury niche might protect the OLs/OPCs from apoptosis. In this study, we confirm the inhibitory effect of PD on the activation of astrocytes and microglia/macrophages. Those effects may contribute to the glial
cell survival, and enhance remyelination together with axon regeneration in turn. In this cellular and molecular context, EGFR inhibitor PD emerged as a cytoprotective agent on OLs lineage owing in part to the inhibition of the secondary damage from astrocytes and microglia. In summary, inhibition of EGFR signaling pathway can reduce tissue damage at both acute and subacute stages of SCI. The therapeutic mechanisms include ameliorating the secondary injury from microglia and astrocytes activation together with regulating growth and differentiation of OLs lineage. Application of EGFR inhibitor after SCI could be a promising approach to promote remyelination, axonal regeneration and functional recovery.
Funding The study was funded by National Natural Science Foundation of China (No. 81470053) and Natural Science Foundation of Shanghai, China (No. 14ZR1435900). Compliance with Ethical Standards Conflict of interest All authors declare that there are no conflicts of interest.
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Cell Mol Neurobiol Ethical approval All procedures performed in the study involving animals were in accordance with the ethical standards of Institutional Animal Care and Use Committee of Nanyang Technological University, Singapore and Xinxiang Medical University, China.
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