Ischemia/Reperfusion-induced neovascularization in the cerebral cortex of the ovine fetus

Abstract

Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.

Figure 1

Representative toluidine blue-stained sections from the sham-control treatment (Non-I/R) (A), ischemia/reperfusion 48 hours (I/R-48) (B), and ischemia/reperfusion 72 hours (I/R-72) (C) groups, taken at regular intervals and utilized to identify similar brain levels and cerebral cortex areas for morphometric analysis. Bars: 5 mm

Figure 2

Confocal images of sheep fetal cerebral cortex double immunostained for glial fibrillary acidic protein (GFAP) and collagen type IV (Coll IV). (A, B) Control sham treatment (Non-I/R) shows healthy-appearing glio-vascular components from the outer, subpial (A), to the deeper (B) cortex layers. Both glia limitans (arrows) and perivascular astrocytes (arrowheads) tightly contact penetrating cerebral cortical microvessels and their branches revealed by Coll IV vascular basal lamina (VBL) and uniformly distributed within the cerebral cortex. (C, D) In ischemia/reperfusion 48 hours (I/R-48) fetal cerebral cortices, reactive astrogliosis involves both glia limitans (arrows) and parenchymal astrocytes, which appear hypertrophic, express high levels of GFAP, and form remarkable perivascular envelopes (arrowheads). (E, F) Apart from an evident astrogliosis, which also include the glia limitans (arrows), the main feature of ischemia/reperfusion 72 hours (I/R-72) cerebral cortices is the number of very small vessels surrounded by their Coll IV VBL and by perivascular astrocytes (arrowheads). Nuclear counterstaining TO-PRO3. Bars: 25 μm

Figure 3

Confocal images of sheep fetal cerebral cortex double immunostained for glial fibrillary acidic protein (GFAP) and collagen type IV (Coll IV). (A–F) Compared with sham-control treatment (Non-I/R) cerebral cortices (A, B), in both ischemia/reperfusion 48 hours (I/R-48) (C, D) and ischemia/reperfusion 72 hours (I/R-72) (E, F) groups, a diffuse astrogliosis is clearly recognizable; hypertrophic astrocytes form perivascular end-feet in contact with a thick Coll IV-enriched vascular basal lamina (VBL) (arrowheads in C–F). Nuclear counterstaining TO-PRO3. Bars: A, C, E = 25 μm; B, D, F = 10 μm

Figure 4

Representative binary images of cerebral cortical fields submitted to morphometric analysis. Collagen type IV (Coll IV)-immunostained microvessels were segmented by an interactive thresholding function and the immunoreactive areas measured and expressed as the percentage of fractional vessel density (mean ± SD). Non-I/R, Control sham treatment; I/R-48, ischemia/reperfusion 48 hours; I/R-72, ischemia/reperfusion 72 hours. Bars: 25 μm.

Figure 5

Quantification of neovascularization in randomly selected areas of sheep fetal cerebral cortex from sham-control treatment (Non-I/R), ischemia/reperfusion 48 hours (I/R-48), and ischemia/reperfusion 72 hours (I/R-72) groups. Vessel density, expressed as the percentage area (mean value ± SD) of fields (total area) segmented on anti-collagen type IV (Coll IV)-stained sections, is significantly increased when I/R-72 brains are compared with control brains (P<0.01) and with the shorter time of reperfusion (I/R-48; p < 0.05). One-way analysis of variance (ANOVA) and Bonferroni Multiple Comparison test.

Figure 6

Confocal images of sheep fetal cerebral cortex double immunostained for glial fibrillary acidic protein (GFAP) and collagen type IV (Coll IV), merge (A, C, E) and single channel (B, D, F) images. (A, B) There is a rare sprouting-like microvessel in the cerebral cortex from ischemia/reperfusion 48 hours (I/R-48) group; the endothelial cell tip shows a long exploring filopodium surrounded by reactive astrocytes (arrowheads). (C–F) Sprouting-like vascular endings (arrowheads) surrounded by a number of seamless-appearing microvessels (asterisks) in ischemia/reperfusion 72 hours (I/R-72). Note that in A–F pericytes are included in the Coll IV-reactive vascular basal lamina (VBL) (arrows). Nuclear counterstaining TO-PRO3. Bars: 10 μm.

Figure 7

Confocal images of ischemia/reperfusion 72 hours (I/R-72) cerebral cortices double immunostained for glial fibrillary acidic protein (GFAP) and collagen type IV (Coll IV) (A) and single stained for Coll IV (B, C, D). (A) Astrogliosis and activated microvessels are common at this time of cerebral cortical reperfusion; there are many endothelial cell nuclei in the wall of a growing microvessel (A, arrows). (B–D) Leading tip-like endothelial cells (arrows) are revealed by the associated Coll IV vascular basal lamina (VBL). Nuclear counterstaining TO-PRO3. Bars: A–C = 10 μm; D = 5 μm

Figure 8

Confocal images of ischemia/reperfusion 72 hours (I/R-72) cerebral cortices double immunostained for glial fibrillary acidic protein (GFAP) and collagen type IV (Coll IV) (A–C) and on Coll IV single channel (D–F). (A, B) Activated microvessels show a thick Coll IV vascular basal lamina (VBL) with embedded pericytes (arrows), and tube-like vessel structures completely ensheathed by perivascular astrocytes (arrowheads). The single channel from these pictures (green; D, E) show pericytes located within the VLM (arrows) and vessel tubes (arrowheads) are clearly recognizable. (C, F) A pair of astrocytes (asterisk in C) extends processes to a filopodia-like protrusion of a pericyte (arrowhead in channel green, F). Bars: 10 μm.

Figure 9

Confocal images of ischemia/reperfusion 72 hours (I/R-72) cerebral cortices double immunostained for glial fibrillary acidic protein (GFAP) and Ki67 (A–D) and collagen type IV (Coll IV) and Ki67 (E, F). (A) Sparse Ki67-reactive astrocytes in the outer cerebral cortical layers (arrows) and a cerebral cortical microvessel showing 2 Ki67-labeled endothelial cells (arrowheads); note the pair of astrocytes or doublets (asterisks); one is located close to the vessel wall and Ki67-reactive endothelial nuclei can be distinguished more clearly in the enlargement (C, asterisk and arrowheads, respectively). (B) A microvessel located deeper within the cerebral cortex ensheathed by Ki67-reactive perivascular astrocytes (arrows) also showing a proliferating endothelial cell (arrowhead); note a pair of Ki67-labeled perivascular cells (asterisks); the one closest to the vessel wall is shown in the enlargement (D; asterisk), along with the Ki67-reactive endothelial cell (arrowhead) and 2 proliferating astrocytes (arrows). (E, F) Cerebral cortical microvessels, whose profile is revealed by Coll IV (asterisk), show Ki67-reactive endothelial cells (arrowheads) and in (E), Ki67-labeled nuclei in the surrounding parenchyma (arrows). Bars: A, B = 25 μm; C, D = 10 μm; E, F = 7.5 μm

Figure 10

The area fractional vessel density (%) of the frontal cerebral cortex plotted on the y-axis against the frontal cerebral cortical concentration of basic fibroblast growth factor (FGF-2/mg total protein on the x-axis) on the x-axis, r = 0.81, n = 8, p = 0.015.