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Intranasal epidermal growth factor treatment rescues neonatal brain injury

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Abstract

There are no clinically relevant treatments available that improve function in the growing population of very preterm infants (less than 32 weeks’ gestation) with neonatal brain injury. Diffuse white matter injury (DWMI) is a common finding in these children and results in chronic neurodevelopmental impairments1,2. As shown recently, failure in oligodendrocyte progenitor cell maturation contributes to DWMI3. We demonstrated previously that the epidermal growth factor receptor (EGFR) has an important role in oligodendrocyte development4. Here we examine whether enhanced EGFR signalling stimulates the endogenous response of EGFR-expressing progenitor cells during a critical period after brain injury, and promotes cellular and behavioural recovery in the developing brain. Using an established mouse model of very preterm brain injury, we demonstrate that selective overexpression of human EGFR in oligodendrocyte lineage cells or the administration of intranasal heparin-binding EGF immediately after injury decreases oligodendroglia death, enhances generation of new oligodendrocytes from progenitor cells and promotes functional recovery. Furthermore, these interventions diminish ultrastructural abnormalities and alleviate behavioural deficits on white-matter-specific paradigms. Inhibition of EGFR signalling with a molecularly targeted agent used for cancer therapy demonstrates that EGFR activation is an important contributor to oligodendrocyte regeneration and functional recovery after DWMI. Thus, our study provides direct evidence that targeting EGFR in oligodendrocyte progenitor cells at a specific time after injury is clinically feasible and potentially applicable to the treatment of premature children with white matter injury.

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Figure 1: Enhanced EGFR expression in oligodendrocyte lineage cells prevents oligodendrocyte and myelin loss, and ultrastructural and behavioural deficits caused by neonatal hypoxia.
Figure 2: EGFR activity is crucial for white matter recovery after neonatal hypoxia.
Figure 3: Intranasal HB-EGF accelerates oligodendrocyte regeneration and promotes cellular recovery in white matter after neonatal hypoxia.
Figure 4: Intranasal HB-EGF promotes ultrastructural, physiological and behavioural recovery in white matter after neonatal hypoxia.

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Acknowledgements

We thank J. Dupree for advice on electron microscopy analysis. We thank L.-J. Chew, J. Corbin and J. Liu for comments on the manuscript. We thank M. McKenna for discussion on metabolic studies. We thank D. Bergles for providing the PDGFαR-CreER mice and D. W. Threadgill for the EGFRfl/fl mice. We thank R. Packer for his support. This work was supported by National Institutes of Health grants: K08NS073793 (J.S.); NSADA K12NS052159 (J.S.); K08NS069815 (S.S.); P01 NS062686 (V.G., T.L.H.); R01NS045702 (V.G.); R01NS056427 (V.G.); P30HD040677 (V.G.); R01MH067528 (F.H.); P30 NS05219 (F.H.); R01MH067528 (F.H.); and the Pioneer Award DP1 OD006850 (T.L.H.). The Childhood Brain Tumor Foundation (J.S.) and the National Brain Tumor Society (J.S.) also provided support.

Author information

Authors and Affiliations

Authors

Contributions

J.S. designed all experiments with V.G. J.S. performed all experiments except electron microscopy and DTI imaging. T.R.H. performed all the Notch analyses. S.S. performed metabolic studies. J.R. and J.S. performed all electrophysiological analyses of CAPs. B.J. and M.R. assisted with experiments. K.S.-B. and T.L.H. performed the electron microscopy studies. D.C., Y.H. and F.H. performed DTI imaging and analysis. R.J.M. performed statistical analyses with J.S. on all behavioural experiments. V.G. supervised the entire project. J.S. and V.G. wrote the manuscript.

Corresponding author

Correspondence to Vittorio Gallo.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Hypoxia results in a significant increase in EGF levels in the white matter.

The white matter was dissected out at P11, P15 and P18 in normoxia (Nx)- and hypoxia (Hyp)-exposed CNP-EGFP (Rep) and CNP-EGFP-hEGFR (Rep-hEGFR) mice. At P11, in both Hyp groups, there was a significant increase in EGF levels, as measured by ELISA. There was no significant difference between the two Hyp groups, indicating that overexpression of EGFR in oligodendrocyte lineage cells does not modify endogenous EGF levels. At P15 and P18, there was no difference between all four groups. All histograms are presented as mean absorption (optical density (OD)) relative to total protein concentration ± s.e.m. **P < 0.05; ***P < 0.01 (P11 and P15, n = 4 mice per group and per age; P18, n = 3 mice per group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons).

Extended Data Figure 2 Enhanced EGFR expression in oligodendrocyte lineage cells prevents oligodendrocyte death and promotes proliferation of OPCs in white matter.

ad, Representative ×40 confocal images of Rep+PI+ cells from normoxia and hypoxia-exposed white matter at P11 in Rep and Rep-hEGFR mice. e, At P11 and P18, hypoxia resulted in a significant increase in the number of oligodendrocyte cells undergoing apoptosis (Rep+Casp3+). Enhanced EGFR expression prevented this increase at all time points, except P60 where no difference was evident. Comparison of the Hyp Rep with the Hyp Rep-hEGFR groups demonstrates that hEGFR in oligodendrocyte lineage cells is protective against apoptosis induced by hypoxia (n = 4 mice per group and per age; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). f, PI was injected intraperitoneally 1 h before mice were killed. A significant increase in the number of Rep+PI+ oligodendrocyte cells indicated membrane disruption contributing to cell death (n = 3 mice per group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). gj, Representative ×40 confocal images of Rep+NG2+ OPCs from normoxia and hypoxia-exposed white matter at P18 in Rep and Rep-hEGFR mice. k, At P11 and P18, Rep-hEGFR Nx mice had more NG2+ OPCs compared with the Rep Nx group. Hypoxia resulted in a significant increase in the number of Rep+NG2+ OPCs in both Rep and Rep-hEGFR groups; however, overexpression of hEGFR did not have an additive effect. l, At P11 and P18, the Rep-hEGFR group had more Rep+Ki67+ cells; however, this did not reach significance (P > 0.05). Hypoxia resulted in enhanced oligodendrocyte-lineage proliferation at P11 and P18, but hEGFR overexpression did not have a significantly additive effect when compared with the Rep Hyp group. k, l, n = 4 mice per group and per age; one-way ANOVA, Bonferroni post-hoc test for individual comparisons. m, More OPCs were in a proliferative state in Rep-hEGFR Nx mice compared with Rep Nx. Hypoxia enhanced OPC proliferation, and overexpression of hEGFR resulted in a significantly additive increase compared to the Rep Hyp group (n = 3 mice per group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). All histograms are presented as means ± s.e.m. Scale bars, 50 μm (ad, gj). *P = 0.05; **P < 0.05; ***P < 0.01.

Extended Data Figure 3 The inclined beam-walking task requires normal subcortical white matter.

ac, This study was performed to test the hypothesis that the inclined beam-walking task is a good assessment of subcortical white matter function. To test this hypothesis, we tested a well-established model of subcortical white matter demyelination induced by bilateral lysolecithin injections in 8-week-old C57BL/6J male and female mice. Animals were tested at 5 days after surgical intervention—which is a time period when demyelination is at its maximum—to determine whether subcortical white matter integrity is important in this behavioural task. Control mice received bilateral injections of normal saline using the same coordinates as the lysolecithin group. a, b, Bilateral demyelination was confirmed after testing by removal of brains and immunohistochemical analysis of corpus callosum. Only mice that had clear bilateral lesions on microscopic examination were included in the behavioural analysis (n = 3 mice were excluded). c, The lysolecithin-injected mice had a marginally significant or very significant increase in average number of foot slips on the 2-cm- and-1-cm-wide inclined beam, respectively (two-tailed Mann–Whitney test, n = 6 per group). d, We wanted to determine whether the performance of Rep (CNP-eGFP) transgenic mice on the inclined beam-walking task was similar to that of C57BL/6 mice (wild type). No difference in performance was evident between the two different lines of male mice. We found that the Rep and wild-type mice performed similarly in either normoxic or hypoxic conditions (Poisson multiple regression analysis; Nx Rep, n = 7; Nx wild type, n = 11; Hyp Rep, n = 8; Hyp wild type, n = 11). All histograms are presented as means ± s.e.m. *P = 0.05; **P < 0.05; ***P < 0.01.

Extended Data Figure 4 Inhibition of EGFR prevents expansion of progenitor cells in the developing white matter and after hypoxia.

ad, Representative ×40 confocal images of subcortical white matter Sox2+ cells, a transcription factor expressed in proliferating multipotential neural progenitor cells. e, Gefitinib, a specific EGFR inhibitor, resulted in a significant decrease in the number of white matter Sox2-expressing cells compared with normoxia vehicle-treated mice. After hypoxia, there was a significant expansion of Sox2+ cells in the white matter compared with the Nx vehicle group; however, this expansion was prevented by gefitinib (Hyp gefitinib) (Hyp vehicle versus Hyp gefitinib, P < 0.01) (n = 4 for each group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). fi, Representative ×40 confocal images of subcortical white matter Ascl1+ cells (Mash1) in the Ascl1–eGFP transgenic mice. Ascl1 is a proneural transcription factor expressed in proliferating multipotential neural progenitor cells. j, Similar to above, gefitinib resulted in a significant decrease in the total number of Ascl1–eGFP+ cells in the white matter compared with normoxia vehicle-treated mice. Hypoxia resulted in a significant expansion in the number of Ascl1–eGFP+ cells, which gefitinib prevented (Hyp vehicle versus Hyp gefitinib, P < 0.05) (n = 4 for each group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). All histograms are presented as means ± s.e.m. **P < 0.05; ***P < 0.01. ad, fi, Scale bars, 50 μm.

Extended Data Figure 5 Intranasal HB-EGF does enter the brain and activates EGFRs in oligodendrocyte lineage cells.

ac, Saline or HB-EGF was administered intranasally once in P11 mice, which were then killed at 1, 5, 15 and 30 min after administration. a, Western blot analysis was performed on microdissected white matter probing for actin, HB-EGF and pEGFR (Tyr 1068 phosphorylation site). In the saline group, no HB-EGF was detected in the white matter and no change in pEGFR was detected. In the mice that received HB-EGF, the HB-EGF protein was detected at 5 min and increased up to 30 min. The pEGFR signal steadily increased at 5, 15 and 30 min after HB-EGF administration. b, c, The line graphs represent relative abundance of protein compared with actin (n = 3 for each time point and condition). Line graphs are presented as means ± s.e.m. d, A normoxia-treated P11 mouse was administered HB-EGF and killed 30 min later. Immunohistochemistry of pEGFR was performed. In the white matter, there were several Rep+pEGFR+DAPI+ cells, indicating that oligodendrocyte lineage cells express activated EGFR (pEGFR). Shown is a ×40 representative image of the white matter demonstrating Rep+pEGFR+DAPI+ cells. e, In this set of experiments, Hyp Rep mice received either intranasal saline or HB-EGF from P11–P14 (Fig. 3b). The subcortical white matter was microdissected at P15 and CNP–eGFP+ cells were FACS purified. Western blot analysis was performed to probe for pEGFR. The western blot demonstrates that a more robust signal for pEGFR was present in the Hyp HB-EGF group, relative to actin (n = 4 for each group of 2–3 pooled brains).

Extended Data Figure 6 Intranasal HB-EGF treatment increases the number of oligodendrocyte lineage cells derived from PDGFαR-expressing OPCs.

a, PDGFαR-CreER;Z/EG transgenic mice were divided into four groups. Saline or HB-EGF was administered intranasally. Intraperitoneal injections of tamoxifen were administered at P12, P13 and P14 in the morning, 1 h before the morning dose of saline/HB-EGF. Mice were killed at P18. be, Representative ×40 confocal images of CC1+ cells (red) derived from PDGFαR-CreER;Z/EG (GFP+) (green) progenitors at P18. fi, Cells derived from PDGFαR-expressing progenitors after hypoxia and after HB-EGF treatment belong to the oligodendrocyte lineage (Olig2+). Representative ×40 confocal images of the subcortical white matter in all four groups at P18. In serial sections from each PDGFαR-CreER;Z/EG mouse, all GFP+ cells in the subcortical white matter co-stained with anti-Olig2+ antibody in all four groups (n = 4 for each group). In all four groups, no GFP+ cells co-stained with anti-GFAP or anti-glutamate/aspartate transporter (GLAST) antibody (data not shown). j, Hypoxia results in a significant increase in the number of GFP+ cells in the white matter and HB-EGF has an additive effect (n = 4 mice per group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). Histograms are presented as means ± s.e.m. bi, Scale bars, 50 μm. **P < 0.05; ***P < 0.01.

Extended Data Figure 7 HB-EGF treatment prevents hypoxia-induced changes in white matter axonal g ratios at P30.

a, b, Scatter plots depicting g ratios versus axon diameter. The lines represent linear fits to pooled data from all mice for each genotype (n = 3 mice per group). a, The scatter plot demonstrates that the Nx saline and Nx HB-EGF groups were similar. b, The scatter plot demonstrates that the Nx HB-EGF and Hyp HB-EGF groups were similar. c, Histogram demonstrating that, at P30, the percentage of myelinated subcortical white matter fibres was significantly decreased in the Hyp saline group. No significant difference was found in the Hyp HB-EGF group. **P < 0.05; ***P < 0.01.

Extended Data Figure 8 Intranasal HB-EGF prevents loss of NAA after hypoxia.

a, 1H-NMR spectroscopy was performed on dissected white matter at P11, P18 and P30. A full-scale representative spectrum is shown where the peak for NAA is at 2.0 p.p.m. The spectrum shown on Fig. 4l is truncated. b, Western blots of aspartoacylase (ASPA), an enzyme found in oligodendrocytes and responsible for hydrolysation of NAA for myelin production in the developing brain. Hypoxia does not result in any significant change in the amount of ASPA present in the white matter at each of the time points listed (P11 and P30, n = 4 for each group and age; P18, n = 5 for each group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons, except P11 unpaired t-test). Histograms are presented as means ± s.e.m.

Extended Data Figure 9 Protocol for late HB-EGF administration used for this study.

In this study, HB-EGF or saline was administered at a later time point. Beginning in the morning of P18, HB-EGF or saline was administered every 12 h until the morning of P21. Intraperitoneal BrdU was administered 1 h before HB-EGF or saline administration, from P18–P21. The inclined beam-walking task was performed at P35. Only Rep mice were used for this study. The histogram is presented in Fig. 4p.

Extended Data Figure 10 Intranasal HB-EGF accelerates oligodendrocyte maturation in the white matter after hypoxia by preventing Notch activation.

a, Microdissected white matter was probed for activated Notch intracellular domain (NICD) and its ligand Delta1. Western blot analysis obtained from microdissected white matter at P11, P14.5 and P18 with actin as a loading control. b, c, Histograms represent quantification of the density of NICD (b) and Delta1 (c) signal normalized to actin. b, At P11 and P14.5, there was a significant increase in the amount of NICD in the Hyp group. No significant difference was evident at P18. The Hyp HB-EGF group had no significant increase at P14.5 compared with the Nx HB-EGF group, and significantly less than the Hyp saline group (n = 4 mice for each group and age; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). c, Delta1 was increased at P14.5 only in the Hyp saline group (P11: n = 4 for each group; P14.5 and P18: n = 5 for each group and age; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). dg, Representative ×40 confocal images of subcortical white matter in the transgenic Notch reporter (TNR) mice, where eGFP is expressed upon activation of Notch effector C-promoter binding factor 1 (CBF1), a downstream transcriptional target of Notch. h, Histogram represents the number of eGFP+Olig2+ cells at P14.5 in the white mater. The Hyp saline group showed a significant increase in eGFP+Olig2+ cells, corresponding to enhanced Notch activation in oligodendrocyte lineage cells. This contributes to delayed maturation of oligodendrocyte lineage cells observed after hypoxia (n = 4 mice per group; one-way ANOVA, Bonferroni post-hoc test for individual comparisons). Histogram is presented as means ± s.e.m. dg, Scale bars, 50 μm. **P < 0.05; ***P < 0.01.

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Scafidi, J., Hammond, T., Scafidi, S. et al. Intranasal epidermal growth factor treatment rescues neonatal brain injury. Nature 506, 230–234 (2014). https://doi.org/10.1038/nature12880

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