Targeting epidermal growth factor receptor to recruit newly generated neuroblasts in cortical brain injuries

Background Neurogenesis is stimulated in the subventricular zone (SVZ) of mice with cortical brain injuries. In most of these injuries, newly generated neuroblasts attempt to migrate toward the injury, accumulating within the corpus callosum not reaching the perilesional area. Methods We use a murine model of mechanical cortical brain injury, in which we perform unilateral cortical injuries in the primary motor cortex of adult male mice. We study neurogenesis in the SVZ and perilesional area at 7 and 14 dpi as well as the expression and concentration of the signaling molecule transforming growth factor alpha (TGF-α) and its receptor the epidermal growth factor (EGFR). We use the EGFR inhibitor Afatinib to promote neurogenesis in brain injuries. Results We show that microglial cells that emerge within the injured area and the SVZ in response to the injury express high levels of TGF-α leading to elevated concentrations of TGF-α in the cerebrospinal fluid. Thus, the number of neuroblasts in the SVZ increases in response to the injury, a large number of these neuroblasts remain immature and proliferate expressing the epidermal growth factor receptor (EGFR) and the proliferation marker Ki67. Restraining TGF-α release with a classical protein kinase C inhibitor reduces the number of these proliferative EGFR+ immature neuroblasts in the SVZ. In accordance, the inhibition of the TGF-α receptor, EGFR promotes migration of neuroblasts toward the injury leading to an elevated number of neuroblasts within the perilesional area. Conclusions Our results indicate that in response to an injury, microglial cells activated within the injury and the SVZ release TGF-α, activating the EGFR present in the neuroblasts membrane inducing their proliferation, delaying maturation and negatively regulating migration. The inactivation of this signaling pathway stimulates neuroblast migration toward the injury and enhances the quantity of neuroblasts within the injured area. These results suggest that these proteins may be used as target molecules to regenerate brain injuries. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-023-04707-1.


Introduction
The discovery of neurogenesis in speci c regions of the adult brain, revealing its capacity to generate new neurons from neural stem cells (NSC), has represented a breakthrough in the understanding of brain plasticity and repair 1,2 .Furthermore, unearthing the fact that neurogenesis is positively regulated in speci c brain regions in response to damage has raised much expectation over the past few years about the opportunity of developing future therapies aimed to regenerate the damaged brain 3 .Physiologically, neurons are replaced in speci c regions of the adult brain.One of these regions is the olfactory bulb (OB), which continuously integrates neuroblasts generated in the subventricular zone (SVZ) 2 .OB neurogenesis is initiated in the SVZ upon activation of neural stem cells (NSC) that enter the cell cycle to generate highly proliferative transit amplifying cells (TAC).These undifferentiated progenitors give rise to neuroblasts.Neuroblasts may undergo cell divisions while they migrate to the OB and differentiate into mature neurons 4,5,6 .This migration of SVZ neuroblasts toward the OB is tightly regulated by signals within the SVZ niche and the OB 7 .
In response to injuries (traumatic, ischemic or other kinds) NSC are activated within the SVZ 8, 9, 10 .As a result, a higher number of neuroblasts can be found in the SVZ of mice with cortical brain injuries 11 .Interestingly, in response to severe ischemic injuries, a subset of these neuroblasts alter their physiological migration route toward the OB and attempt to migrate toward the damaged region 12,13,14,15 .However, neuroblast migration from the SVZ to a non-extensive focal cortical injury rarely occurs 16 .SVZ cells have been observed migrating toward brain injuries only in speci c injured regions such as the striatum and few reports show migration toward the injured cortex, in response to extensive ischemic injuries 17 .In most cases, the majority of the cells that initiate a migratory pathway toward the injury stay within the corpus callosum (CC), barely reaching the perilesional area 16,18,19,20 .In ammatory cues released by immune cells in response to the damage 21 may be one of the main factors involved in the ine cacy of neurogenic niches to repair brain injuries.Thus, understanding the signaling molecules involved in regulating neurogenesis in response to injuries might highlight the role of molecules involved in brain injury repair.In here, we have studied the role of transforming growth factor alpha (TGF-α) and its receptor, the epidermal growth factor (EGF) receptor (EGFR).TGF-α is a signaling molecule previously reported as participating in the modulation of neuroblast migration from the SVZ toward the OB 22,23 .TGF-α is a member of family, structurally homologous to EGF that binds to EGFR 24 .This receptor is expressed in activated NSC and TAC, as well as in immature neuroblasts.The activity of this receptor and its associated pathways stimulates the proliferation of these cells 25,26,27 .TGF-α is released to the extracellular medium in a mechanism dependent on the metalloprotease ADAM17 and facilitated by kinases of the protein kinase C (PKC) family, particularly the classical PKCα 28, 29 .We hypothesize that mechanical cortical injuries upregulate the expression of TGF-α in the injured cortex and the SVZ leading to higher levels of this ligand.This stimulates the generation of neuroblasts and promotes their proliferation while it impairs neuroblast maturation and migration.Therefore, the inhibition of EGFR with after a cortical injury may result in the migration of SVZ cells toward the injury resulting in neuroblast enrichment within the injured area.Since the use of EGFR inhibitor drugs has been approved to treat disease such as non-small cell lung cancer (NSCLC) testing the effect of EGFR inhibitors may be useful to identify pharmacological treatments to regenerate cortical brain injuries.

Materials
Most products were purchased from Merck Life Science.The classical PKC inhibitor Gö6976 was purchased from Calbiochem (Millipore, Billerica, MA, USA).The classical PKC inhibitor was dissolved in dimethyl sulfoxide (DMSO) and diluted to a nal concentration of 0.16 µM in phosphate-buffered saline (PBS) before the administration.The EGFR inhibitor Afatinib (MedChemExpress, USA) was dissolved in DMSO and diluted to a nal concentration of 1 µM in PBS.

Animal Subjects
CD1 male mice were used throughout this study.Animals were housed under controlled conditions of temperature (21-23°C) and light (LD 12:12) with free access to food (AO4 standard maintenance diet, SAFE, Épinay-sur-Orge, France) and water.Care and handling of animals were performed according to the Guidelines of the European Union Council (2010/63/EU), and the Spanish regulations (65/2012 and RD53/2013) for the use of laboratory animals.All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals 30,31 .
The number of animals used in each experiment was determined based on previous studies 32,33,34 .
Adult male mice were randomized during the rst week after birth by cross-fostering and used when they became two months old.The protocol used has been authorized by the Ethics Committee of the "Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible de la Junta de Andalucía", in Spain with the approval numbers 04/03/2020/033 y 10/03/2020/039.All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals 30,31 .

Experimental Design
Mice received controlled mechanical injuries in the primary motor cortex while anesthesized with a cocktail of 100 mg/kg ketamine and 20 mg/kg xylazine For the migration studies, mice were injected with lentiviral vectors prior to the injury in the same surgical act.Once injured, the different treatments were administered intranasally as we describe in the paragraphs below.Upon the completion of the treatments were completed, the mice anesthesized with a cocktail of 100 mg/kg ketamine and 20 mg/kg xylazine and CSF was extracted as explained below, then a dose of Dolethal® (Ventoquinol, Lure, France) containing a lethal 50 mg dose of pentobarbital to euthanized the animals was applied followed by either brain perfusion (for histological postmortem studies) or brain extraction (for molecular biology studies).See description of the different procedures below.

Mechanical Cortical Brain Lesions
Controlled unilateral mechanical cortical brain injuries were performed in the primary motor cortex of the right brain hemisphere of anesthetized mice.A cocktail of 100 mg/kg ketamine and 20 mg/kg xylazine was used as anesthetic and injected intraperitoneally to mice (n = 5-6 per group as indicated in gure legends).Anesthetized mice were unilaterally lesioned in the right hemisphere of the primary motor cortex.Using a stereotaxic frame (Kopf Instrument), mice were craniotomized with a mechanical drill at + 1.1 mm rostral and + 1.5 mm lateral to Bregma.Thereafter, a controlled mechanical lesion was performed in the underlying primary motor cortex using a manual drill (0.7 mm diameter).This drill was allowed to penetrate 1 mm below the bone surface.Mice were injured and placed into a controlled cage for 7 or 14 days post injury (dpi) depending on the treatment and experimental design.Lesions were performed unilaterally; the injured hemisphere was considered the ipsilateral side, while the intact hemisphere was considered the contralateral hemisphere and was used as a control.This procedure was previously stablished by our research group and has been used elsewhere 11,18,19,20 .

Intranasal Administration
The classical PKC inhibitor (Gö6976) and Afatinib were delivered intranasally as previously described while the animal was placed in a standing position with an extended neck 35,36,37 .18 µL of 1 µM Afaitinib, 0.16 µM Gö6976 or diluent (vehicle, containing 0.4% DMSO) was delivered over both nasal cavities alternating 3 µL/each using a micropipette.To ensure all uid was inhaled, mice were maintained in the mentioned stained position for 10 additional seconds.Mice were coded, treatment (control or treatment) was assigned randomly to code numbers and applied.In addition, blind quanti cations were performed.

Cerebrospinal Fluid Extraction
Cerebrospinal uid (CSF) collection was performed as described by Lim et al., 2018 38 .Mice were anesthetized as described above and placed prone on the stereotaxic instrument.Muscles were moved to the side and dura mater over the cisterna magna was exposed.The capillary tube was placed and inserted into the cisterna magna through the dura mater, lateral to the arteria dorsalis spinalis.Finally, the CSF was collected.

Concentration Of Tgf-α In Csf
TGF-α was measured in the CSF using commercial ELISA kits, MBS2508394, (MyBioSourse, Inc, San Diego, CA), following the manufacturer's instructions.CSF was centrifugated for 20 min at 1000xg and 4º C; then supernatant was collected.Blanks (diluent only) were included in each independent determination.Blanks were subtracted from measurements before comparisons were made.

Rna Isolation, Reverse Transcription And Real-time Quantitative Pcr
For RT-qPCR analysis, RNA was isolated from the SVZ; intact SVZ were processed for RNA extraction using the TRIzol™ (Cat.15596026, Invitrogen, Carlsbad, CA, USA), separation method, following the manufacturer's instructions and resuspended in puri ed nuclease-free water.RNA was quanti ed using a BioTek's Synergy™ Mx uorimeter (BioTek Instruments, Inc, Winooski, VT, USA).cDNA was prepared from 500 ng RNA using iScript™ cDNA Synthesis Kit (Cat.1708890,Bio-Rad Laboratories Inc, Hercules, CA, USA) on a Techne Genius thermal cycler (Techne Ltd., Cambridge, UK).The 15 µl RT-qPCR reaction mix contained 7.5 µl 2X iTaq™ Universal SYBR® Green Supermix (Cat.1725122, Bio-Rad Laboratories Inc, Hercules, CA, USA), 10 nmol of both the forward and the reverse primers, and 1 µl of the sample.The PCR thermal pro le included 40 cycles of denaturation at 95°C for 10 sec, an annealing temperature according to each set of primers for 15 sec, and extension at 72°C for 20 sec, followed by a melting curve analysis.Each sample was analyzed in triplicate.The mRNA level of rRNA18S was used as internal control.
Oligonucleotides primers used in this study were designed by BLAST and were obtained from Merck (Madrid, Spain).Primer sequences (5´-3´) for detecting expression of mouse mRNA were the following: for TGF-α, FW: CCAGATTCCCACACTCAGT, RW: GGAGGTCTGCATGCTCACA; for EGFR, FW: CCAGACAGACGACGGGTCA, RW: GCTCTGGCTCTCCGGGATTA.

Svz Lentiviral Transductions And Afatinib Treatment
Mice were mechanically injured in the primary motor cortex and were injected with a lentiviral vector expressing ZsGreen in the lateral ventricle (LV).The lentiviral construct was produced by us as described in previous reports 19 .The virus titer of the lentivirus solution was 40 x 10 3 TU/ml and 1 µL of the lentiviral solution was injected within the LV, to perform the lentivirus administration a small trepanation was made 0.8 mm lateral to Bregma and a needle of 100-µL Hamilton Neuros syringe (0.108 mm internal diameter) was introduced 2.4 mm below the brain surface.This lentivirus only induced the expression of ZsGreen in infected cells and accounted for any effect caused by transduction.The EGFR inhibitor Afatinib (1 µM) was administrated during 14 days after the injury and the lentiviral administration procedure.

Brain Processing And Immunohistochemistry
At the end of treatments, mice were deeply anesthetized with Dolethal® (Ventoquinol, Lure, France) containing a lethal 50 mg dose of pentobarbital and perfused with 4% paraformaldehyde via ascending aorta.After perfusion, brains were sliced into 30 µm sections using a cryotome.Immunostaining was performed as previously described 18,33,39,40 .See antibodies in Supplementary tables 1, 2.

Quanti cation Of Neurogenesis In Brain Sections
After perfusion, mouse brains were codded and blind quanti cation was performed.SVZ cells positive for the different markers were estimated as previously described 19 .Positive cells were counted throughout the entire lateral or laterodorsal walls of the lateral ventricles in every fth section; analyzing 14-16 sections per brain under confocal microscopy (Zeiss LSM 900 Airyscan 2).The SVZ cell quanti cation was done in 30 µm thick serial coronal sections including, in the rostrocaudal axis in relation to Bregma, from + 1.54 to -0.94 mm.Confocal imaging was taken every 2 µm in the Z-plane using 20X, 40X, or 63X objectives.Cell density was calculated for each section relative to the SVZ volume (mm 3 ) and averaged for each animal as reported previously 41 .Both SVZ quanti cation was performed using the ImageJ software.
To analyze cell number in the perilesional area, 3-5 sections containing the cortical injury were selected.
Positive cells of each marker were counted in each section including 200 µm-wide band of the adjacent injured border tissue.Cell density (number of cells counted divided by injured area and by section thickness) was calculated for each section, and averaged for each animal.

Statistical Analysis
All data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology 42 .The Student t-test for paired samples was used to compare ipsilateral vs. contralateral brain hemispheres.One-way ANOVA, followed by post-hoc Bonferroni test was used to compare the groups under study.IBM SPSS statistics 22 software was used for all statistical analysis.Differences were considered signi cant values of p < 0.05.Generally, sample size used in statistical analysis was n = 4-6, and was chosen based on previous works 19,33 .

Results
Expression of TGF-α is altered in the SVZ and injured cortex in response to a mechanical cortical injury Unilateral mechanical cortical injuries in the primary motor cortex of mice were performed and the levels of the mRNAs encoding TGF-α and EGFR were analyzed 7 dpi and 14 (Fig. 1A) by qRT PCR in the ipsilateral and contralateral SVZ and cortex (Fig. 1C).In the injured cortex, the mRNA encoding TGF-α and EGFR increased by 3-and 1.5-fold respectively in the ipsilateral injured side in comparison with the contralateral side 7 dpi (Fig. 1C).14 dpi, the elevated levels of TGF-α mRNA in the injured cortex remained elevated whereas EGFR levels declined to reach those found in the contralateral side and in control mice (Fig. 1C).In the SVZ, the levels of TGF-α mRNA increased by 2-fold 7dpi and remained elevated 14 dpi.(Fig. 1D).Interestingly, in the SVZ, no changes were observed in the expression of EGFR 7 dpi (Fig. 1D) but a 2-fold increase in EGFR mRNA was found 14 dpi.In agreement, the concentration of TGF-α in the CSF of injured mice 7 dpi increased by nearly 3-fold (Fig. 1B) these levels were maintained 14 dpi.

Injury-induced Increase In The Number Of Tgf-α Microglial Cells In The Svz And Cortex
A 4-fold increase in the number of cells labelled with TGF-α was found in the injured cortex (Fig. 1E, F).Accordingly, a 2.5-fold increase in the TGF-α burden was found 7 dpi in the ipsilateral SVZ in comparison with the contralateral SVZ (Fig. 1G, H).Interestingly a dramatical increase in the number of Iba1 + /TGF-α + was observed in the ipsilateral cortex of injured mice.A smaller but signi cant increase was also found in the SVZ.The posterior analysis of these results indicated that the phenotype of the cells expressing TGFα in the injured cortex (Fig. 2A, B) and the SVZ (Fig. 2C, D) was mainly Iba1 + microglial cells.

The Number Of Immature Svz Neuroblasts Increases In Response To A Cortical Injury
The number of cells expressing EGFR in the ipsilateral SVZ 7 dpi increased by 2-fold (Fig. 3A, B) and this was concomitant with a 2-fold enrichment in the number of Ki67 + cells (Fig. 3C, D).In addition, the pool of neuroblasts increased by 1.5-fold in the ipsilateral SVZ 7 dpi (Fig. 4A, B), particularly, EGFR + neuroblasts increased dramatically by 6-fold in the ipsilateral SVZ (Fig. 4A, C) and a 2-fold increase in the number of cycling Ki67 + /DCX + neuroblasts was found 7 dpi (Fig. 4A, D).

Reduction Of Tgf-α Release Impairs The Proliferative Response In Svz Neuroblasts
The release of TGF-α was then inhibited using the classical protein kinase C (cPKC) inhibitor (Gö6976) since cPKC activation stimulates the ADAM17 mediated release of TGF-α in vitro 29 .Mice were injured and treated for 7 days with intranasal administrations of the cPKC inhibitor Gö6976 or vehicle.The concentration of TGF-α in the CSF was analyzed at 7dpi as well as the number of cycling cells and neuroblasts.As shown in Fig. 5, Gö6976 treatment reduces the concentration of TGF-α in the CSF of treated mice by 6-fold (Fig. 5E), concomitantly, the increase in the number of cycling cells in the ipsilateral SVZ of injured mice was not found upon treatment with Gö6976 (Fig. 5A, B) and the number of proliferating neuroblasts not only was not elevated in the ipsilateral SVZ but it was reduced by 2-fold in comparison with the contralateral side (Fig. 5A, D).The burden of DCX labelling was not affected by the use of the inhibitor (Fig. 5A, C).These results suggested a role for TGF-α on stimulating the proliferation of neuroblasts and maintaining the immature phenotype of neuroblasts within the SVZ.

Inhibition Of Egfr Facilitates Neuroblasts Enrichment Within The Injured Area
Finally, in order to demonstrate the role of TGF-α on neuroblast migration, in another set of experiments, we injected a lentiviral vector expressing ZsGreen in the lateral ventricle of adult mice and on the same day a mechanical injury was performed in the motor cortex.Lentiviral vectors were produce within the group as described previously.Then mice were treated with intranasal administrations of Afatinib for 14 days and mice were sacri ced 14 dpi (Fig. 6A-D).In control untreated mice, cells containing ZsGreen were found within the perilesional area, a small number of them being DCX + cells.However, in mice treated with Afatinib, a 5-fold increase in the number of DCX + / ZsGreen + cells was found within the perilesional area.In the SVZ, no differences were observed in the DCX + burden or the number of DCX + / ZsGreen + cells (Supplementary Fig. 1).

Migration Of Neuroblasts Toward The Injury Is Induced By The Inhibition Of Egfr
Interestingly, chains of migrating DCX + cells, some of them expressing ZsGreen, were observed at 14 dpi that had crossed the corpus callosum from the SVZ toward the cortex.These cells were only observed in mice treated with Afatinib (Fig. 7B; see right panels for magni cation) and not in control mice (Fig. 7A).

Discussion
Brain injuries of different origins stimulate neurogenesis in the SVZ resulting in a higher number of newly generated neuroblasts that attempt to migrate toward the injury but do not reach the perilesional area 16,19 not contributing to the regeneration of the injured region.Failure of neuroblasts to migrate toward the injury has been reported to be a consequence of the presence or absence of signaling molecules that affect neuroblast migration 22,43 .Understanding the cues that modulate neuroblast enrichment in the SVZ as well as migration of these cells toward the injury might lead to the generation of pharmacological drugs aimed to repair brain injuries.We show in here that injuries induce the expression of TGF-a in both the SVZ and the cortical injured area resulting in elevated levels of TGF-a in the CSF.We show that TGFa is mainly expressed in microglial cells that appear in both regions in response to the injury.As a consequence, neuroblasts remain in an immature proliferating state not being able to migrate toward the injury.Inhibition of the TGF-a receptor EGFR facilitates migration of neuroblasts from the SVZ toward the injury resulting in an enrichment of neuroblasts within the perilesional area.
Previous studies have demonstrated that brain damaged caused by ischemia or by traumatic injury, stimulate neurogenesis in neurogenic niches or in the damaged area (reviewed in Nemirovich-Danchenko and Khodanovich 2019 44 ) depending on the extent of the injury and the affected area.Thus, the proliferation of neural stem cells and other progenitors is stimulated in the SVZ in response to ischemic injuries and some authors have shown that these progenitors migrate from the SVZ toward the damaged striatum producing neurons that integrate into existing circuits 12,45,46 .However, although other studies show the presence of a low number of newly formed neurons in the damaged cortex post injury 47 , others have failed to demonstrate cortical neurogenesis post injury 11,12,19 .A few years ago, the work of Sundholm-peters et al. showed that in controlled cortical aspiration lesions a subset of neuroblasts emigrate from the SVZ dorsally into the corpus callosum.Interestingly, a higher number of these cells are observed within the corpus callosum whereas no neuroblast reached the injured cortex 16 .Additional reports show that a signi cant number of neuroblasts can be found with the injury upon application of BDNF 43 , the inhibition of ADAM17 19 or the treatment with PKC-activating compounds involved in neuregulin release 20 .
In here, we aimed to discover new mechanisms involved in the response of the brain to cortical injuries, we initially showed that an overexpression of TGF-a mRNA is found in the ipsilateral SVZ of injured mice and in the perilesional area.This elevated expression was concomitant with an augmented concentration in soluble TGF-a in the CSF of the injured mice and with an increased number of cells expressing TGF-a in the ipsilateral SVZ and injured cortex as soon as 7 dpi.The phenotype of these TGF-a + cells was mainly microglial cells in both the SVZ and injured cortex.Our results show that in response to the injury, Iba1 + microglial cells appear in the perilesional area and the SVZ.These microglial cells expressed TGF-a.Similar results have previously been found by Goings et al.Using aspiration lesions of the cerebral cortex, they observe the presence of Iba1 + microglial cells in the cortex, striatum and corpus callosum of injured mice 48 .TGF-a expression has been found in injured regions in other cellular phenotypes such as Nestin + cells and astroglial (GFAP + ) cells 11 .
The elevated expression of TGF-a is accompanied by an overexpression of its receptor EGFR.Recent reports show that TGF-α expression is signi cantly increased in microglia/macrophages and neurons after ischemia as well as its receptor, EGFR 49 .In order to understand whether this TGF-a played a role in the regulation of post injury neurogenesis, we started by evaluating neurogenesis in the SVZ, where EGFR stimulates the activation of NSC, the proliferation of TAC 25,26,27 and the proliferation of immature neuroblasts 22 .We have found that in the ipsilateral SVZ of injured mice an increased number of Ki67 + proliferating cells is found together with an elevated number of EGFR + cells.This results indicate that, according to previous reports, 11,14,18 , a proliferative response to the injury is found in the SVZ of injured mice that is concomitant with the elevated number of EGFR + cells, with the upregulation of the EGFR and TGF-a mRNAs and with an elevated concentration of TGF-a in the CSF.An upregulation of the mRNA of other EGFR ligands such as EGF has previously been shown around the lesion and extending into the SVZ 16 supporting the idea of the role that EGFR initiated pathway plays in neurogenesis post-injury.This is the rst time in which an increased concentration of TGF-a in the CSF is described in response to a cortical injury.Interestingly, a higher number of neuroblasts within the ipsilateral SVZ of injured mice showed an immature phenotype in response to the injury, expressing EGFR and the cell cycle marker Ki67 suggesting that the stimulation of the EGFR-initiated pathway may impair maturation.In accordance, a previous report shows that immature EGFR + proliferating neuroblasts are found in the SVZ of mice treated with TGF-a and in these neuroblasts migration toward the OB is impaired 22 .In this occasion, we did not see any neuroblasts within the perilesional area 14 dpi.Inhibition of TGF-a release by using a classical protein kinase C (cPKC) inhibitor, dramatically reduced the concentration of TGF-a in the CSF as well as a reduction in the number of immature neuroblasts.The cPKC-dependent release of TGF-a has previously been demonstrated in cell cultures using time lapse images and uorescent probes 28,29,50 .In here we observe a similar effect of the cPKC inhibitor in vivo when administered intranasally, that is accompanied by a reduction in the total number of neuroblasts and in the percentage of immature neuroblasts.
Finally, in order to demonstrate the role of TGF-a and EGFR on the migration of these neuroblasts toward brain injuries we have treated adult mice with mechanical cortical injuries with the EGFR inhibitor Afatinib; using a lentiviral vector that expressed the green uorescent marker ZsGreen, we show that the inhibition of EGFR facilitates the enrichment of ZsGreen + neuroblast around the perilesional area.
Imaging techniques show that these neuroblasts migrate in chains from the SVZ to the injured cortex.Interestingly, a previous report from our laboratory shows that if PKC activity is inhibited locally within the injury an enrichment in neuroblasts can be observed but these neuroblasts do not seem to be SVZmigrating cells 18 .Thus, it is possible that TGF-a exerts its antimigratory effect on SVZ neuroblasts and that Afatinib administered intranasally is reaching the SVZ and exerting its effect on SVZ neuroblasts.This result highlights the role of the signaling pathway TGF-a/EGFR as inhibitor of neuroblast migration.This idea agrees a previous work, which show that an excess TGF-a impair emigration of neuroblast toward the OB 22 .
Afatinib is a tyrosine kinase inhibitor, which use was approved by the FDA in 2013 51 for the treatment of patients with locally advanced or metastatic NSCLC with activating EGFR mutations who are EGFR tyrosine kinase inhibitor naïve 52 .We have chosen Afatinib to inhibit EGFR in this work, however, we know that other receptors of the ErbB family could be lightly inhibited by Afatinib.Alternatively, other EGFR inhibitors could be of use such as erlotinib or ge tinib, and testing a battery of these inhibitors may be interesting in future studies.Thus, as summarized in gure 8, we show in here that in response to the injury a large number of microglial cells expressing TGF-a emerge within the SVZ and the injured area.TGF-a is released from these cells and activates the EGFR present in the neuroblast membrane inducing its proliferation, delaying maturation and probably impeding migration.The use of the EGFR inhibitor Afatinib stimulates neuroblast migration and enrichment in neuroblasts of the injured area.These neuroblasts could probably differentiate into mature cortical neurons if the treatment persisted.Our results suggest that TGF-a or EGFR may be used as targets to regenerate brain injuries.The use of this or other EGFR inhibitors upon mechanical cortical injuries may result in the repair of the injured brain tissue.

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