Intraventricular hemorrhage in premature infants: mechanism of disease

Abstract

Intraventricular hemorrhage (IVH) is a major complication of prematurity. IVH typically initiates in the germinal matrix, which is a richly vascularized collection of neuronal-glial precursor cells in the developing brain. The etiology of IVH is multifactorial and is primarily attributed to the intrinsic fragility of the germinal matrix vasculature and the disturbance in the cerebral blood flow (CBF). Although this review broadly describes the pathogenesis of IVH, the main focus is on the recent development in molecular mechanisms that elucidates the fragility of the germinal matrix vasculature. The microvasculature of the germinal matrix is frail because of an abundance of angiogenic blood vessels that exhibit paucity of pericytes, immaturity of basal lamina, and deficiency of glial fibrillary acidic protein (GFAP) in the ensheathing astrocytes endfeet. High VEGF and angiopoietin-2 levels activate a rapid angiogenesis in the germinal matrix. The elevation of these growth factors may be ascribed to a relative hypoxia of the germinal matrix perhaps resulting from high metabolic activity and oxygen consumption of the neural progenitor cells. Hence, the rapid stabilization of the angiogenic vessels and the restoration of normal CBF on the first day of life are potential strategies to prevent IVH in premature infants.

Figures

Figure 1. Morphology of germinal matrix

A) Representative cresyl violet staining of coronal section of the right-sided cerebral hemisphere of a 20-week fetus. Note cortical plate (arrowhead), white matter (arrow with 2 pointers), germinal matrix (arrow head), caudate nucleus (asterisk), and lateral ventricle (indicated by “v”). Germinal matrix (violet staining) surrounds the whole ventricle, but is most conspicuous on the head of caudate nucleus. Scale bar, 0.5 cm. B) Representative immunofluorescence of cryosection from germinal matrix of a 24 week premature infant labeled with DAPI (blue), GFAP (green) and CD34 (red). Note: germinal matrix is highly vascular (vascular endothelium in red) and enriched with GFAP (+) glial cells (green). C) Coronal brain section was double labeled with doublecortin (red, labels neuronal precursors) and CD34 (green, labels endothelium) specific antibodies. Note doublecortin (+) neuronal precursor cells are abundantly present in the germinal matrix. Scale bar; 50 (B) and 20µm (C). D) Schematic drawing of the blood brain barrier in cross section showing endothelium, endothelial tight junction, basal lamina, pericyte and astrocyte endfeet.

Figure 2. Germinal matrix is deficient in fibronectin and pericytes

A) Representative immunofluorescence of cryosection from germinal matrix and white matter of a 24-week premature infant labeled with fibronectin (red) and CD34 (green) specific antibody. Fibronectin is strongly expressed in the white matter (arrowheads) whereas it is weakly expressed in the germinal matrix (arrows). B) Cryosection from germinal matrix and white matter of a 24-week premature infant was double-labeled with CD34 (endothelium, red) and NG2 (pericyte, green) specific antibody. NG2 positive pericytes are intimately associated with the outer endothelial surface. The coverage of the endothelia by NG2 positive astrocytes is interrupted and incomplete in the germinal matrix (arrow). In contrast, in the white matter, the vascular coverage by pericytes is continuous and complete (arrowhead). Scale bar; 20µm.

Figure 3. Rapid endothelial proliferation in the hypoxic germinal matrix

A) Representative immunofluorescence of cryosection from germinal matrix and white matter of a 21-week fetus labeled with Ki67 (proliferating nuclei, green) and CD34 (red) specific antibody. Note abundant Ki67 positive nuclei in the germinal matrix (arrowhead), but only few in the white matter. CD34 stained vessels with Ki67 signal (arrowhead) indicate endothelial proliferation in the germinal matrix. Left panel also shows orthogonal views in x–z and y–z planes of a composite stack of a series of images acquired by a confocal microscope. Right panel shows CD34 positive blood vessel with RBC in the lumen (arrow), but no Ki67 positive signal in the blood vessel. Scale bar; 20µm. B) A premature rabbit pup (E29) was injected with hypoxyprobe (60 mg/kg dose) immediately after birth and pup was euthanized after 2 hours. Hypoxyprobe is metabolized by nitro-reductase enzymes in case of low pO2 level of to form hypoxyprobe chemical adducts. Frozen sections were stained with hypoxyprobe monoclonal antibody to detect hypoxyprobe adducts. Note: intense immuno-staining (double headed arrow) in the periventricular germinal matrix and weak immunoreactivities in the white matter. V, ventricle. Scale bar; 20µm.

Figure 4. Mechanisms underlying fragility of germinal matrix vasculature

Hypoxic germinal matrix induces VEGF and angiopoieitn-2 expression. These growth factors trigger angiogenesis. The nascent vessels of the germinal matrix exhibit paucity of pericytes and deficiency of fibronectin in immature basal lamina. In addition, astrocyte endfeet in the germinal matrix vasculature display diminished expression of GFAP. These factors contribute to the fragility of the germinal matrix vasculature. Black arrow in the schematic of neovasculature shows angiogenic blood vessel (grey, endothelium and black, pericyte) lacking pericytes.