Early microglial colonization of the human forebrain and possible involvement in periventricular white-matter injury of preterm infants

Abstract

Amoeboid microglial subpopulations visualized by antibodies against ionized calcium-binding adapter molecule 1, CD68, and CD45 enter the forebrain starting at 4.5 postovulatory or gestational weeks (gw). They penetrate the telencephalon and diencephalon via the meninges, choroid plexus, and ventricular zone. Early colonization by amoeboid microglia-macrophages is first restricted to the white matter, where these cells migrate and accumulate in patches at the junctions of white-matter pathways, such as the three junctions that the internal capsule makes with the thalamocortical projection, external capsule and cerebral peduncle, respectively. In the cerebral cortex anlage, migration is mainly radial and tangential towards the immature white matter, subplate layer, and cortical plate, whereas pial cells populate the prospective layer I. A second wave of microglial cells penetrates the brain via the vascular route at about 12-13 gw and remains confined to the white matter. Two main findings deserve emphasis. First, microglia accumulate at 10-12 gw at the cortical plate-subplate junction, where the first synapses are detected. Second, microglia accumulate in restricted laminar bands, most notably around 19-30 gw, at the axonal crossroads in the white matter (semiovale centre) rostrally, extending caudally in the immature white matter to the visual radiations. This accumulation of proliferating microglia is located at the site of white-matter injury in premature neonates. The spatiotemporal organization of microglia in the immature white and grey matter suggests that these cells may play active roles in developmental processes such as axonal guidance, synaptogenesis, and neurodevelopmental apoptosis as well as in injuries to the developing brain, in particular in the periventricular white-matter injury of preterm infants.

Fig. 1

The three main morphological aspects of microglial cells. From Rezaie & Male (1999).

Fig. 2

(A) Lateral sagittal section through a 5.5-gestational week (gw) human embryo showing ionized calcium-binding adapter molecule 1 (Iba1)-positive cells issued from the choroid plexus (ChP) and constituting patch 1a in the eminentia thalami (EM/ET). Patch 2 is contiguous to the ditelencephalic fissure (arrows). Grey areas vary in colour depending on the Iba1-positive cell density. The box corresponds to C, where patches of microglial cells are indicated by asterisks. (B,C) Iba1 (black)-CD34 (brown) double labelling and counterstaining with neutral red. (B) Sagittal section through an 8-gw embryo [level indicated in Fig. 2G in Monier et al. (2006)]. Iba1-positive patches 1a, 1b, and 2 are visible near the ditelencephalic fissure (arrow), patch 1a near the presumptive anlage of the internal capsule, and patch 3b at the presumptive anlage of the external capsule. (D,E) High magnification at the level of ChP and ET shown in A. Fluorescent double labelling for Iba1 (green) and CD45 (red) (D) and for Iba1 (green) and CD68 (red) (E) showing co-localization (yellow). Note the preferential CD68 labelling deeper in the EM parenchyma (asterisk in E) compared with the preponderant CD45 labelling in the ChP (asterisk in D). ca, cortical anlagen; Dien, diencephalon; GE, ganglionic eminence; post, posterior; Dth, dorsal thalamus; VZ, ventricular zone; V, ventricle. Scale bars: B, 500 μm; C, 100 μm; D and E, 25 μm. Adapted from Figs 1 and 2 in Monier et al. (2006).

Fig. 3

(A–C,E) Ionized calcium-binding adapter molecule 1 (Iba1) (black)-CD34 (brown) double labelling. (A,B) Sagittal section through a 4.5-gestational week (gw) embryo at low (B) and high (A, at the level of the asterisk in B) magnification. In A, Iba1-positive cells are located in the ventricle (V) (green arrowhead) and along the pial surface (green arrow). ant, anterior part of the central nervous system; M, meninges; VZ, ventricular zone. (C) Sagittal cerebral wall and choroid plexus (ChP) at 9 gw; intermediate Iba1-positive cells accumulate under the cortical plate (CP) and in the intermediate zone (IZ), displaying virtually no contiguity with CD34-positive vessel walls. The ChP exhibits numerous amoeboid Iba1-positive microglia. One positive cell close to the ventricular surface (arrow) is visible next to other positive cells in the VZ. MZ, marginal zone. (D) At 12 gw, in the IZ and subplate, Iba1-positive cells exhibit elongated processes that are orientated radially (arrowheads) and tangentially (arrow), consistent with a migratory phenotype. (E) In the subplate at 23.5 gw, Iba1-positive perivascular cells (green arrowhead) are first detected along a CD34-positive vessel wall next to ramified Iba1-positive cells (long arrow). (F–J) At 19 gw, CD45-positive cells are scattered throughout the cingulate cortex (F); they have an intermediate star-like phenotype in the lower CP (G), a tangential and/or radial orientation in the deep subplate (SP) (H), and multiple long multi-directionally orientated processes in the IZ (I). These cells display an intermediate phenotype in the subventricular zone (SVZ) and they are numerous and amoeboid in the VZ (J). Magnification: A, 200×; B, 16×; C, 50×; D,G–J, 400×; E, 100×; F, 20×. Adapted from Figs 1 and 2 in Monier et al. (2007).

Fig. 4

(A–C) Fetuses aged 9 gestational weeks (gw) (A,B) and 10.5 gw (C). (A) Ionized calcium-binding adapter molecule 1 (Iba1)-CD34 double labelling showing almost no contact between microglial cells (black) and vessels (brown) at 9 gw in the internal capsule (A), whereas one vessel in the white matter contains numerous contiguous microglial cells at 10.5 gw (C, long arrow). Note in C that other blood vessels located in the grey matter on the right in the thalamic anlage medial to the internal capsule, indicated by two arrowheads, do not contain any contiguous positive microglia (arrowheads). (B) Numerous proliferating MIB1 (clone of monoclonal mouse anti-human Ki-67 antigen) (black)-Iba1 (brown) double-labelled microglia in patch 3b at 9 gw (arrows). (D) Coronal sections in a 12-gw embryo; CD45-positive microglia at the border of the third ventricle (IIIrdV) (arrowheads), in the ventricular zone of the IIIrdV, and in the hypothalamic area. (E) Cluster of Iba1-positive cells (4a) in the medial septum at 14.5 gw. AC, anterior commissure; lv, lateral ventricle. (F) Cluster 2 near the optic tract (OT) at 16 gw. (G–J) Double-labelled microglial cells at the same magnification in patch 3b. Cells positive for CD68 (black)-Iba1 (brown) (G,I) and for CD45 (black)-Iba1 (brown) (H,J) in a 13-gw fetus (G,H) and a 23.5-gw fetus (I,J). Note the larger size of amoeboid microglia earlier during development (G,H), especially for CD45-positive cells (H). CD45 labelling is more diffuse in the later-stage specimen (J), whereas CD68 labelling is dot-like (I). Scale bars: A,C, 100 μm; B,G–J, 20 μm; E, 100 μm; F, 300 μm. Adapted from Figs 5–7 in Monier et al. (2006).

Fig. 5

Coronal anterior (A) and posterior (C) ionized calcium-binding adapter molecule 1 (Iba1)-labelled sections through a 10.5-gestational week (gw) human fetus. Grey areas vary in colour according to the density of Iba1-positive cells. (B,D) Iba1-labelled sections at the level of the corresponding boxes in A and C. Cluster 1a is in the thalamus (Th) extending along the dorsal Th (A,C), cluster 1b in the ventricular zone of the ganglionic eminence (GE) zone, and cluster 3a in the genu of the internal capsule (IC) (B). Patches 3a, 3b, and 3c are located at the junction of the IC with the Th, external capsule (EC), and cerebral peduncle (CP), respectively. Patch 2 surrounds the optic tract (OT). ChP, Choroid plexus; VZ, ventricular zone. Scale bars: B and C, 500 μm. From Fig. 3 in Monier et al. (2006).

Fig. 6

(A–C) Contiguous sections through the dorsal frontal cortex of a 10.5-gestational week (gw) embryo showing intermediate ionized calcium-binding adapter molecule 1 (Iba1)-positive (A) and CD68-positive (B) cells aligned at the junction between the cortical plate (CP) and subplate (SP) (arrowheads). (C) Tyrosine hydroxylase-immunolabelled contiguous section showing catecholaminergic cortical axonal afferents toward the cerebral cortex, restricted at this developmental stage to the intermediate zone and SP (Zecevic & Verney, 1995). (D) On a contiguous section, numerous double-labelled Iba1 (brown)-MIB1 (clone of monoclonal mouse anti-human Ki-67 antigen) (black) cells (arrowheads), i.e. proliferating microglia–macrophages, in the SP, most notably at the junction with the CP. (E,F) Iba1 labelling of a coronal dorso-frontal section through the foramen of Monro showing a cluster of Iba1-positive cells at axonal crossroads at 22 gw (E, long arrow) and 23.5 gw (F, long arrow). IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone; V, ventricle. (G,H) High magnification of Iba1-positive (G) and CD68-positive (H) intermediate microglial cells in the cluster located in the dorsal occipital cortex of a 19-gw specimen. (I,J) Double labelling for Iba-1 (black, arrows) and glial fibrillary acidic protein (brown) at the crossroads (C2). (I,J) Note, at the same magnification, the high density of Iba1-positive microglia (black) at 25 gw, contrasting with the sparse distribution of these cells at 32 gw. CC, corpus callosum; IZ, intermediate zone; SVZ, subventricular zone; V, ventricle; VZ, ventricular zone. Scale bars: A–C, 50 μm; E, 3 mm; F, 500 μm; D,G–J, 20 μm. Adapted from Figs 2 and 3 in Monier et al. (2007) and additional data.

Fig. 7

Cases with no neuropathological lesions [A, 20 gestational weeks (gw); C, 25 gw] and with periventricular white-matter injury (PWMI) (B, 22 gw; D, 26 gw). Fluorescent double labelling for ionized calcium-binding adapter molecule 1 (Iba1) (green) and N-methyl-D-aspartate receptor 1 (NMDAr1) (red) (A,B) and for Iba1 (green) and inductible nitric oxide synthase (iNOS) (red) (C,D) showing co-localization in the amoeboid–intermediate microglia located in the C2 crossroads in the cases with PWMI. In B, note the double-labelled macrophages (yellow, long arrows) and the non-double-labelled (i.e. less activated) microglia (short arrows). Other cells exhibit dot-like red NMDAr1 labelling (asterisk in B) and iNOS labelling (short arrow in D). Scale bars: A–D, 20 μm.