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Identification and prevention of heterotopias in mouse neocortical neural cell migration incurred by surgical damages during utero electroporation procedures Identification and prevention of heterotopias in mouse neocortical neural cell migration incurred...
Wang, Bolin; Ji, Liting; Bishayee, Kausik; Li, Changyu; Huh, Sung-Oh
2020-03-03 00:00:00
ANIMAL CELLS AND SYSTEMS 2020, VOL. 24, NO. 2, 114–123 https://doi.org/10.1080/19768354.2020.1737225 Identification and prevention of heterotopias in mouse neocortical neural cell migration incurred by surgical damages during utero electroporation procedures a a b a b Bolin Wang *, Liting Ji *, Kausik Bishayee , Changyu Li and Sung-Oh Huh a b School of Pharmacy, Zhejiang Chinese Medical University, Hangzhou, People’s Republic of China; Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallym University,Chuncheon, South Korea ABSTRACT ARTICLE HISTORY Received 21 February 2020 In utero electroporation (IUE) is a useful technique for gene delivery in embryonic mouse brain. IUE Revised 24 February 2020 technique is used to investigate the mammalian brain development in vivo. However, according to Accepted 25 February 2020 recent studies, IUE methodology has some limitations like the formation of artificial ectopias and heterotopias at the micro-injection site. Thus far, the artificial heterotopias generated by physical KEYWORDS trauma during IUE are rarely reported. Here, we reported the artificial heterotopias and ectopias In utero electroporation; generated from surgical damages of micropipette in detail, and moreover, we described the micro-injection; artificial protocol to avoid these phenotypes. For the experimental purpose, we transferred empty heterotopias and ectopias; plasmids (pCAGIG-GFP) with green fluorescent-labelled protein into the cortical cortex by IUE layer marker Ctip2 and TBR1 and then compared the structure of the cortex region between the injected and un-injected cerebral hemispheres. The coronary section showed that ectopias and heterotopias were appeared on imperfect-injected brains, and layer maker staining, which including Ctip2 and TBR1 and laminin, can differentiate the physical damage, revealing the neurons in artificial ectopic and heterotopic area were not properly arranged. Moreover, premature differentiation of neurons in ectopias and heterotopias were observed. To avoid heterotopias and ectopias, we carefully manipulated the method of IUE application. Thus, this study might be helpful for the in utero electroporator to distinguish the artificial ectopias and heterotopias that caused by the physical injury by microneedle and the ways to avoid those undesirable circumstances. Introduction delivered or transfected into the cerebral cortex, dience- phalon, hindbrain, and spinal cord region (David et al. Neuronal disorders have a significant impact on the new- 2014; Nicole et al. 2018; Watanabe et al. 2018) and borns, faulty genetic distribution can contribute to used for the evaluation of the phenotypic changes that different disease related to brain development. There- are relevant to the disease pathology (Cwetsch et al. fore, it is important to understand the role of a specific 2018). But IUE technique has different drawbacks that gene during the neuro-development process. Therefore, could lead to different unexpected false or imitation gene manipulation in the embryonic mouse brain is results, such as surgical damages to cause artificial important for understanding how the genetic disturb- ectopias and heterotopias. In this paper, we will discuss ances contribute to either disease etiology or develop- different issues with IUE during the injection procedure. mental disorders. Traditional gene manipulation techniques like gene- In utero electroporation (IUE) is a useful technique targeting technologies using embryonic stem (ES) cells, for gene manipulation in a particular part of the brain zinc-finger nucleases (ZFNs) technology and transcrip- (Nishimura et al. 2012; Hong et al. 2018; Hu et al. 2018; tion activator-like effector nucleases (TALENs) technology Kim et al. 2018). The expression vectors are microinjected were used to study neuro-development from ages (Pav- into the embryonic brain ventricle, and transfect letich and Pabo 1991; Li et al. 1992; Capecchi 2005; the neuronal precursor cells with the DNA by square- Moscou and Bogdanove 2009; Higashijima et al. 2017; wave electric pulses (Niwa et al. 2010; Youngpearse Boch et al. 2009). The major disadvantages of these et al. 2010). Using this technique, the DNA can be methods are such as limitation in transfection efficiency CONTACT Changyu Li lm159@sina.com School of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang, People’s Republic of China; Sung-Oh Huh s0huh@hallym.ac.kr Department of Pharmacology, College of Medicine, Institute of Natural Medicine, Hallym University, Chuncheon 24252, Gangwon-Do, South Korea *Authors have equally contributed. © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. NEUROBIOLOGY & PHYSIOLOGY ANIMAL CELLS AND SYSTEMS 115 Table 1. Advantages of in utero electroporation Advantages Details References High efficiency, low No significant increase in cell death has been detected after Saito (2006), Mizutani and Saito (2005) cytotoxicity electroporation Simple and quick procedure In vivo electroporation for gene transfer into more than 10 embryos can be Saito (2006) carried out within 30 min Multiple gene targeting Multiple genes on different plasmids can be simultaneously transfected Saito and Nakatsuji (2001) into the same cells Region and time-specific gene Only the side of the ventricle that is close to the anode is transfected Saito and Nakatsuji (2001), Saba et al. (2003), targeting Cwetsch et al. (2018) and specio-temporal gene manipulation, anduncon- (blood vessels in brain marker) (Xu et al. 2019), Nestin trolled or non-specified transfection. IUE has several (progenitor and radial glia (RG) marker) (Halbach 2007), advantages over the traditional techniques like higher Reelin (telencephalon marginal zone marker) (Goffinet transfection efficiency, lower cytotoxicity, time-efficient, 2017), KI-67 (proliferation marker) (Menon et al. 2019), and multiple plasmids can be simultaneously transfected BrdU (proliferation and DNA synthesis marker) (Halbach into the same cells (Saito 2006; Cwetsch et al. 2018). IUE 2007) to identify and understand the defects forms due requires neither complex surgery nor stereotaxic appar- to surgical damages during IUE. atus, so now, IUE is widely used to understand the mech- anism of brain development (Carrel et al. 2015; Broix et al. Materials and methods 2018). The detail of the advantages of IUE is described in Animals and housing Table 1. However, in IUE, improper injection method can Pregnant mice of C57BL/6N strain were used in this obtain an unwanted or false outcome that can confuse study for IUE. The mice were purchased from Orient researchers. Therefore, every result obtained by IUE Bio (Seongnam, South Korea). All the experiments should carefully be evaluated for the formation of were approved by the Institutional Animal Care and pseudo-heterotopia or pseudo-ectopia-like structures. Use Committee (IACUC) of Hallym University, South Heterotopia is clump-like structures in the ventricular Korea (approval number: Hallym 2015-7). Fetuses at or periventricular zone and forms due to faulty migration E13.5–E17.5 were used for the experimental purpose. of the neurons during development (Ishii et al. 2015; According to Orient Bio’s mating schedule, the Manent et al. 2009). The similar structural deformities vaginal plug at noon of the day was defined as could be formed by injury from the mishandling of the embryonic day 0.5 (E0.5). The mice were housed in needle. The first instance pseudo-heterotopia was docu- optimum conditions (12 h light/day cycle with 22 ± 2° mented by Rosen et al., in 1995, he induced heterotopias C and 50 ± 10% humidity) and mice feeds were and ectopias in the cerebral cortex of newborn mice procured from a commercial food company (Purina through inserting a hypodermic needle into developing Inc., Seongnam, Korea). cerebral cortex (Rosen et al. 1995). Micropipette needle can also induce physical trauma on the cortex which Preparation of plasmid DNA glass capillary would disturb the process of neuronal migration and can result in pseudo-heterotopia and pseudo-ectopia- The pCAGIG plasmid vector containing ampicillin selec- like structures. tion marker were grown in Escherichia coli- DH5a strain. In this article, we described different aspects of IUE The plasmid DNA was isolated from Luria Bertani (LB) that can cause unnatural outcomes. While the artificial broth culture with ampicillin by using Qiagen EndoFree® heterotopias generated by surgical procedures in IUE Plasmid Maxi Kit. DH5a strain was grown in standard LB are rarely reported until now. Here, we report the artifi- medium to a cell density of ∼3–4×10 cells/ml. DNA was cial heterotopias generated from physical injuries by extracted by following the manufacturer’s protocol and micropipette in detail and described the measures to the DNA was precipitated by isopropanol to the eluted avoid the artificial heterotopias. We performed IUE with DNA. Mix and centrifuge immediately at 15,000 g for pCAGIG-GFP plasmid to identify the transfected 30 min at 4°C and the eluted DNA was washed with neurons in the ventricles. Later, the cortex was stained endotoxin-free 70% ethanol at room temperature. with different cellular markers like Tuj1 (neuronal differ- Before the in vivo electroporation, the DNA was dilated entiation marker) (Halbach 2007), Ctip2 (cortex layer 5 to a final concentration of 2 µg/µl in 0.1% fast green and 6 marker) (Leyva and López 2013), TBR1 (cortex (Sigma) solution and TE buffer (10 mM Tris base, 1 mM deep-layer marker) (Englund et al. 2005), Laminin EDTA solution, pH 8.0). G1 glass capillary (Narishige 116 B. WANG ET AL. Scientific Instrument Lab, Tokyo, Japan) was used for the washed with OCT solution to remove the excess injection. The sharp end on the glass capillary for the sucrose–PBS solution and embedded in OCT. The injection was made using a micropipette puller (Nar- coronal sections (10 µm) from brains were sliced using ishige, Model-PC10). Then the capillary end was the cryo-section machine. pinched off with forceps to make the tip with 20– For immunostaining, the antigen retrieval process was 30 µm diameter. performed by heating the slides in citrate buffer (10 mM, pH 6.0) at 95°C for 5–10 min. The samples were blocked in 5% donkey serum in PBS plus 0.1% Triton-X100 (PBST) Preparation for surgery solution. The sections were subsequently incubated with anti-BrdU (Jackson Immunorearch) (1:400), anti-KI67 Pregnant mice were anesthetized using Isoflurane – Ifran (Abcam) (1:100), anti-GFP (Abcam) (1:100), anti-Tuj1, (Hana Pharm. Co. Ltd., Kyonggi-do, Korea) with a con- anti-Ctip2 (Abcam) (1:500), anti-TBR1 (Abcam) (1:500), stant supply of oxygen and nitrogen gas mixture. The anti-Laminin (Sigma) (1:1000), anti-Nestin (DSHB) hair from the abdomen was removed by using a razor (1:400), and anti-Reelin (DSHB) (1:400) for overnight at blade and 83% molecular grade ethanol, later the 4°C in a humid chamber. Afterwards treatment with sec- shaved skin region was sponged with the povidone- ondary antibody (conjugated with fluorescence) was iodine topical solution to remove any contamination. carried out for 2 h at room temperature. A total of 15 The incision was made at the abdominal midline with embryos were used for the study of differentiation and fine scissors and then all uterine horns were carefully proliferation staining. Fluorescent images were acquired pulled out onto a 37°C sterilized pre-warmed phos- with a laser-scanning confocal microscope (LSM710) phate-buffered saline (PBS)-moistened autoclaved (Zeiss, Oberkochen, Germany). cotton gauze, the uterus was kept moist with PBS during the surgical period. Result In utero electroporation Formation of ectopias and heterotopias at The expression vector-pCAGIG containing green fluor- micropipette puncture region at the micro- escent protein (GFP) was used for electroporation. injection site Pregnant mice of different stages were anesthetized To visualize the ectopia and heterotopia formation by in using 4% isoflurane. After surgery, plasmid DNA utero injection, the GFP-positive injected brains were sec- (2 mg/ml) in PBS containing 0.01% fast green stain tioned (Figure 1(A–C)). The neuronal cells are stained (Sigma–Aldrich, St. Louis, USA) was microinjected with green color and nucleus was stained with blue through the uterine wall into the lateral ventricles of color. The heterotopia-like lump structure was identified target embryos. Using Tweezertrodes (5 mm diameter) in low magnified brain slices and marked with a white (BTX/Harvard Biosciences, Holliston, USA) across the arrow (Figure 1(D)). On the other hand, we also identified embryo’s brain through the uterus, electroporation ectopia-like structures in the injected brains and marked was performed by discharging 5 pluses of the 45 V with a yellow line, but in the control set, these types of for 50 ms duration and 950 ms interval. The intact structures were absent (Figure 1(D)). The improperly uterus was then put back to the abdomen and the injected brains showed pseudo-ectopia and heteroto- abdominal cut was carefully and aseptically sutured pia-like lumps, but the lump-like structures was absent back. The mice were allowed to recover in pre- in the properly handled control set, and the ectopias warmed cages. The electroporated embryos were and heterotopias in ventricles may result from physical then allowed to develop in the pregnant mice. The injury by micropipette instead of the specific plasmid. transfected GFP-positive embryo brains were shorted for the further experimental process. Induction of premature differentiation at Immunohistochemistry and identification of micropipette puncture region ectopias and heterotopia To understand the molecular etiology of the pseudo- The extracted brain or whole skull, depending on the ectopic or heterotopic mouse brain, we stained them embryonic stage, immersed in 4% paraformaldehyde with β-III-tubulin (Tuj1), a neuron-specificdifferentiation (PFA) at 4°C for 2 h for fixation. Then the brains were marker. In the control mouse brain, the ventricular stored in PBS containing 30% sucrose at 4°C-overnight zone (VZ) region was Tuj1-negative, suggesting that for the hardening process. Later, the brains were the progenitor cells were in dividing the state. But in ANIMAL CELLS AND SYSTEMS 117 Figure 1. In utero electroporation with green-plasmid and identification of ectopias and heterotopias. (A) The embryonic mouse brains at E13.5 stage were injected with GFP-conjugated plasmid (pCAGIG) in 0.1% fast green solution and then transfected by electropora- tion. (B) The pCAGIG electroporated brains were harvested at E15.5 stage (48 h post-electroporation) and examined under Research Macro Zoom fluorescence system (Olympus DP72, Kanazawa-shi, Japan) for green fluorescence. (C) The rostral part of a coronal section from the injected brain was sectioned and then stained with DAPI (indicated white line from B was the magnified section that represents in C). (D) The harvested brains were sectioned and stained with DAPI to identify the Ectopia and Heterotopias. The Ectopia was indicated with white arrowheads and heterotopias were indicated by a yellow dashed line. Scale bar of the image is 100 μm. heterotopias in VZ, we found that the region was Tuj1- pattern (Figure 3). The disruption of the layers was positive, implying that the induction of premature differ- caused by the microneedle fractures. entiation of the neurons in the proliferative zone Later, we stained the cortex with laminin and nestin (Figure 2). The induction of early or premature differen- which stains basal lamina and redial glial cells, respect- tiation could be one marker for the micropipette- ively. The Laminin staining of the basal lamina of CP mediated damage. revealed that the control set has an intact and smooth structure, whereas that of the micropipette puncture region was of the convex structure and was broken Identification of damage cortex at micropipette also. We stained the RG cells with nestin, the radial puncture region glial cells in control extend long processes that terminate with their endfeet at the pial basement membrane, while It is necessary to identify the damage cortex region of the the radial glial cells in micropipette puncture region developing brain by the micropipette puncture. In the arranged irregularly and bulged out of the pial surface control set, the GFP-positive neurons are migrated into (Figure 3). the intermediate zone (IMZ) and cortical plate (CP) To identify the rupture of the telencephalic marginal region, the neuronal morphological transition at IMZ zone, we stained the cortex with reelin. Reelin stains was prominent. Alternatively, in the ectopic cortex the outer marginal layer of the cortex. In the control region, the distribution of migrating neurons at IMZ brains, the reelin stained a uniform outer layer but with and CP region was not as uniform as the control brains ectopia, reelin staining showed the same tendency that and observed morphological transition at IMZ was neurons and other cells disrupt the reelin and over minimum (Figure 3). Therefore, to understand the distri- migrate out of the pial surface (Figure 3). bution of the neurons at the cortex, we stained the To identify whether the ectopic brains were consisting cortex regions with different layer markers. First, we of proliferating cells, we stained the cortex samples with stained with ctip2 and TBR1 to stain different layers. In two proliferation markers, BrdU and KI67. In the control the control brain cortex, both the layers were distributed tissue, the proliferating cells were at IMZ and ventricular evenly as an indicator of normal cortical structure, but in zone/subventricular zone (VZ/SVZ) area. While compar- the ectopic brain, at the micropipette puncture region, ing with the ectopic brains, there were no significant the layer pattern was changed into a convex curve 118 B. WANG ET AL. Figure 2. Differentiation status of neurons at the ectopic and heterotopic region. The embryonic mouse brains at E13.5 stage were injected GFP-conjugated plasmids and harvested at E 15.5 stage. DAPI (blue) and Tuj1 (white) staining were performed to stain nucleus and differentiated neurons. The ectopia was marked white arrowheads and heterotopias were by a yellow dashed line. The white line was marked for the boundary layer of undifferentiated neuronal region as per the control set. The Tuj1-positive differentiated neuronal population was markedly increased in the ectopic and heterotopic cortical section. The scale bar was 50 μm. changes in the number of proliferating cells, but the The solution to avoid artificial ectopias and structure of the proliferating zone was changed with heterotopias bulged out structure. There was a significant increase in the number of KI67-positive cells in the upper layer In this section, we will be discussing the solutions to region in the ectopic brains, implying the leakiness or avoid the formation of ectopias and heterotopias in the disruption of the neuronal layers (Figure 4). cortical region of the developing brain. ANIMAL CELLS AND SYSTEMS 119 Figure 3. Deformation of cortical layers in section cortex. The embryonic mouse brains at E13.5 stage were injected GFP-conjugated plasmids and harvested at E15.5 stage. (First panel) The neuronal migration was observed by GFP staining (green) in control and ectopic cortical sections. (Second panel) The transfected brain sections were stained with layer markers like Ctip2 (red) and TBR1 (green) and counterstained with the nuclear stain DAPI (blue). (Third panel) The transfected brain sections were stained with basal lamina marker laminin (red) and radial glial cell marker nestin (blue). (Fourth panel) The transfected brain sections were stained with telencephalon marginal zone marker Reelin (red). The scale bar was 50 μm. Figure 4. Proliferation status of neurons in the ectopic region. The embryonic mouse brains of E13.5 stage were injected GFP-conju- gated plasmids and harvested at E15.5 stage. The cortical slices were stained with antibody for GFP (green), KI67 (red), BrdU (blue) and DAPI (gray). GFP-positive neurons at the ectopic site were over-migrated when compared with control section. KI67 and BrdU-positive neurons were found at the higher IMZ region of the ectopic cortex but were absent in control. White dashed lines were used to marked different zones of the cortex (CP, IMZ, and VZ/SVZ). The scale bars were 100 μm for the first panel and 50 μm for rest of the panels. (a) Precautions to follow for cortical injections to step is important for handling the embryos and for avoid artificial ectopias and heterotopias identification of the proper injecting site. Different precautious steps need to be followed during (iii) When the embryo was set up to be perfectly micro-injection in the embryo brain to avoid artificial oriented for micro-injection, then it needs to be ectopias and heterotopias. pierced the uterine wall with glass micropipette at (i) The glass micropipettes quality is essential to an angle of 45° to the uterine wall. reduce artificial ectopia and heterotopia formation. (iv) It is important to identify the rostral end of the gap between cortical hemispheres and the injection (ii) The uterus needs to be held in a way that ventricles could be visualized easily. For proper injection, this penetration should be of ∼1 mm. 120 B. WANG ET AL. (v) One microlitre of DNA-fast green solution needs to be (b) Formation of hippocampus and identification of injected into the ventricle before placing the suitable stage for IUE electric pulse. The proper injection fills the Another major solution we provided in this study is to ventricles with a blue color solution. The optimum transfer plasmids into the hippocampus. In order to pulse rate is described in Table 2 for plasmid DNA identify the suitable stage to operate IUE for the embryo- transfection. nic hippocampus, we stained hippocampus at different embryonic stages by immunohistochemistry. The results showed that hippocampus primordium was formed at E13.5 (Figure 5(A–C)), which only consisted Table 2. Optimal voltages for different embryonic stages of in of stratified periventricular neuroepithelial cells, and vivo electroporation therefore, it is premature for electroporation. At E14.5, Surviving Embryonic stage Voltage (V) embryos (%) GFP+ embryos (%) cornu ammonis has ultimately formed, the neuroepithe- E12.5 35 >95 98 lial cells layer was thickened, and the intermediate layer E12.5 45 < 10 – was formed (Figure 5(D–F)). At E15.5, dentate gyrus E13.5 45 >95 >98 E14.5 45 ∼100 >98 formed, and dentate gyrus folded with cornu ammonis E15.5 45 ∼100 >98 which promote the formation of hippocampal fissure E16.5 45 ∼100 >98 (Figure 5(G–I)). We found that the E14.5 brains were NB: E12.5–E14.5 embryos were generally used for cortical injection; and E14.5–E16.5 embryos for hippocampal injection. the most suitable for hippocampal transfection by IUE. Figure 5. Coronal sections of the embryonic brain at different stages. (A–C) Hippocampus at E13.5 stage, (D–F) hippocampus at E14.5 stage, (G–I) hippocampus at E15.5 stage, and (J–L) hippocampus at E16.5 stage. The white arrows indicate the hippocampus at different stages. The scale bar was 200 μm. ANIMAL CELLS AND SYSTEMS 121 Figure 6. Transfection in the hippocampus region of the embryonic brain. (A) Combinations of different positions of electrodes at the coronal plane for transfection. (B) Transfected regions at the coronal plane resulting from different combinations of electrodes. (C) Com- binations of different positions of electrodes at the sagittal plane for transfection. (D) Transfected regions at the sagittal plane resulting from different combinations of electrodes. (E) The hippocampus transfected with RFP and GFP vectors simultaneously. Two kinds of plasmids with RFP and GFP were transferred into hippocampus at E14.5 stage by IUE, and red fluorescence protein and green fluor- escence proteins were expressed in the hippocampus region. The scale bar was 100 μm. The hippocampus is a three-dimensional structure, so the micropipette puncture region that may produce an the position of the electrode appears to be particularly ectopias and heterotopia-like structures in the later important to determine the area of electroporation, stages of the development. Adaptation of these simple and we optimized here the electrode position to maxi- identification methods can distinguish between the mize the electroporation area. In Figure 6(A), the 1 real ectopias and heterotopia structure that created by to 3 represents the position of the positive pole of the elec- the wound healing process of the needle puncture. trode, and 1–3 represents the position of the negative The tissue integrity was lost in the process of pseudo- ectopia and heterotopia formation. The needle punctu- pole. On the coronal section, electroporation region differed from the combination of electrodeposition, red zone was seen to swell out at the periphery and at and the combination of position-1 of the positive pole the ventricle zone of the cortex and the heterotopia and ectopia bulges could also be measured. To our and position-3 of the negative pole was optimal as it covered most of the hippocampus region (Figure 6(B)). knowledge, this DAPI staining would be a simple And the best combination for the sagittal section was primary observation that can be helpful to identify these little structural deformities in developmental position-3 of the positive pole and position-1 of the negative pole, which also covered most of the hippo- brains. In normal cases, the outer layered cortex used campus on the sagittal section (Figure 6(D)). The hippo- to be tuj1-positive (Halbach 2007), so the neuronal differ- entiation process in the brain could be measured by tuj1 campus was harvested at E18.5 and the sections of hippocampus showed that RFP and GFP could be staining. The improper differentiation was observed in detected expression simultaneously (Figure 6(E)). the pseudo-heterotopia, and ectopia containing the brain. We observed an early differentiation induction at the damaged sites of brain. This early differentiation Discussion with the DAPI-bulges are a prominent marker for the pseudo-heterotopia and ectopia containing the brain The IUE has plenty of beneficiary effect; it also has limit- (Figure 2). ations like surgical damages, transient transfection that To understand the distribution of neurons and their produces false results. We described here the protocol migration process, the morphological transition of the and safety measures for IUE and how surgical damage neurons at the IMZ region is important (Buchsbaum can cause artificial ectopias and heterotopia in the devel- and Cappello 2019). The newborn neurons undergo for oping cortex. We defined different markers to identify 122 B. WANG ET AL. accomplish it. All authors reviewed the manuscript. The work inside out pattern to reach at the CP. Therfore, the IMZ is was funded by the grant from Hallym University Research an important area, where the neurons undergo morpho- Fund, South Korea: [Grant Number HRF-201905-008]. logical transitions. Then later, the bipolar-shaped neurons migrate upwards with the help of radial glial cells. For the brains with ectopia and heterotopia, we Disclosure statement found the neuron’s distribution in the cortex was not uni- No potential conflict of interest was reported by the author(s). formed. Later, we observed the structural integrity of the cortex was disturbed in the ectopic and heterotopic cortex. The marginal layer, the upper layer and Funding the deep layer of the cortex were ruptured; also, overmi- The work was funded by the grant from Hallym University gration of the neurons was also observed in the ectopic Research Fund, South Korea: [Grant Number HRF-201905-008]. brains (Figure 3). While comparing with the ectopic brains, there were no significant changes in the number of proliferating cells, but the structure of the pro- ORCID liferating zone was changed with a bulged out erection Sung-Oh Huh http://orcid.org/0000-0002-6019-6450 in the surgically damaged brains. There was a significant increase in the number of KI67-positive cells at the upper layer region in the ectopic brains, implying the leakiness References or disruption of the neuronal layers. 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