EphrinB3 restricts endogenous neural stem cell migration after traumatic brain injury

Abstract

Traumatic brain injury (TBI) leads to a series of pathological events that can have profound influences on motor, sensory and cognitive functions. Conversely, TBI can also stimulate neural stem/progenitor cell proliferation leading to increased numbers of neuroblasts migrating outside their restrictive neurogenic zone to areas of damage in support of tissue integrity. Unfortunately, the factors that regulate migration are poorly understood. Here, we examine whether ephrinB3 functions to restrict neuroblasts from migrating outside the subventricular zone (SVZ) and rostral migratory stream (RMS). We have previously shown that ephrinB3 is expressed in tissues surrounding these regions, including the overlying corpus callosum (CC), and is reduced after controlled cortical impact (CCI) injury. Our current study takes advantage of ephrinB3 knockout mice to examine the influences of ephrinB3 on neuroblast migration into CC and cortex tissues after CCI injury. Both injury and/or ephrinB3 deficiency led to increased neuroblast numbers and enhanced migration outside the SVZ/RMS zones. Application of soluble ephrinB3-Fc molecules reduced neuroblast migration into the CC after injury and limited neuroblast chain migration in cultured SVZ explants. Our findings suggest that ephrinB3 expression in tissues surrounding neurogenic regions functions to restrict neuroblast migration outside the RMS by limiting chain migration.

Keywords: EphrinB3; Migration; Neuroblast; Traumatic brain injury.

Fig. 1

EphrinB3 and CCI injury alter neuroblast migration in the subventricular zone (SVZ). The SVZ was dissected from non-injured (a,b) and CCI injured (c,d) wild type (WT) (a,c) and ephrinB3^−/− (b,d) mice, and labeled with anti-DCX (red) and anti-CD31 (green) antibodies. In non-injured WT, neuroblasts can be seen predominantly in thick streams (arrowheads) across the dorsal aspect of the SVZ (a). High magnification insets (a′,a″). CCI injury leads to a mixture of tight (arrowheads) and broader streams (arrows) (c). High magnification insets (c′,c″). In ephrinB3^−/− mice, neuroblasts are mainly observed in broad streams (b) and little change after CCI injury (d). High magnification insets (b′,b″,d′,d″).

Fig. 2

EphrinB3 increases neuroblast migration outside the SVZ and RMS. Immunolabeled sagittal brain section from a WT mouse showing anti-DCX labeled neuroblasts (red) in RMS, CC and perilesional cortex 3 days after CCI injury, while anti-CD31 (green) labeled vessels were used for tissue referencing (a). Stereological cell counts of the RMS (b), rostral CC (c), caudal CC (d) and peri-lesional cortex (e) show a rostral-to-caudal gradient of neuroblasts from the rostral CC towards the caudal CC and injury site. Increased neuroblasts numbers were observed in the rostral CC (c), caudal CC (d) and perilesional cortex (e) in ephrinB3^−/− as compared to WT mice. High-magnification images of neuroblasts in the rostral CC (f), caudal CC (g) and perilesional cortex (h). CC, corpus callosum; DG, dentate gyrus; Hipp, Hippocampus; LV, lateral ventricle; OB, olfactory bulb; RMS, rostral migratory stream; SVZ, subventricular zone. *p < 0.05, **p < 0.01 as compared with their respective non-injured controls.

Fig. 3

EphrinB3-Fc infusion reduces neuroblast migration into the overlying CC at 2 days following cannula injury. Immunolabeled sagittal brain section from a WT mouse shows anti-DCX labeled neuroblasts (red) in the RMS, CC and subcortical tissues at 2 days post-injury, while anti-CD31 (green) labeled vessels were used for tissue referencing (a). High-magnification inset of neuroblasts migrating from RMS to CC and subcortical tissues (arrowheads depict neuroblast chains) (a′). Stereology shows increased numbers of neuroblasts in the RMS (b) and CC (c) in Fc-control WT mice as compared with non-injured WT mice. Infusion of ephrinB3-Fc reduced neuroblast numbers in the CC of WT mice (c). EphrinB3^−/− mice show increased chain (d) and isolated cell (e) migration in the rostral CC as compared with WT mice. Infusion of clustered ephrinB3-Fc reduced the number of neuroblast chains/clusters but increased the number of isolated neuroblasts. CC, corpus callosum; RMS, rostral migratory stream; SVZ, subventricular zone. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with their respective non-injured controls; #p < 0.05 as compared with their respective Fc controls.

Fig. 4

Application of ephrinB3 regulates neuroblast chain migration from cultured SVZ explants. Brightfield (a,c,e) and DCX-immunolabeled (b,d,f) SVZ explants show migrating neuroblast in basal (a,b) conditions, unclustered (Uncl.) ephrinB3-Fc (c,d) to block Eph signaling, and clustered (Cl.) ephrinB3-Fc (e,f) to activate Eph signaling. The extent of chain migration and single cell migration were measured using a semi-quantitative scale: 0 = no outgrowth; 1 ≤ 10 chains/cells; 2 = 10–50 chains/cells; 3 = 50–100 chains/cells; 4 = extensive growth. The area of explant outgrowth was measured and expressed as a ratio of outgrowth area (i.e. explant size). Graphs show increased numbers of chains migrating out of the explants after treatment with Uncl. ephrinB3-Fc (g), while isolated cells were increased after treating with Cl. ephrinB3-Fc (h). Both Cl. and Uncl. ephrinB3-Fc increased the total outgrowth of the explants (i). Results in all graphs show mean ± SEM of 39–48 explants per treatment; *p < 0.05 compared to basal conditions.

Fig. 5

EphrinB3 is expressed in the human control and TBI brains. Expression of ephrinB3 in uninjured (n = 2) or traumatized (n = 7) human brain samples as detected by Western blotting. The traumatized samples were grouped into tissue collection between 3 and 24 h (acute; n= 6) or >5 days (122 h; n = 1). No differences were observed between acute TBI samples and uninjured controls; however, in the one sample collected from a patient 122 h after injury a reduced level of ephrinB3 was observed despite a high level of expression of β-actin in this sample.