Morphometric assessment of collagen accumulation in germinal matrix vessels of premature human neonates

Germinal matrix haemorrhage in premature neonates is commonly attributed to vascular immaturity, possibly related to an abbreviated process of angiogenesis. Terminal steps in the progression of angiogenesis are the formation of a subendothelial basal lamina containing collagen IV and an extracellular matrix containing collagens I and III. Immature vessels would predictably be deficient in these collagen subtypes. We analysed germinal matrix (GM), cortical, and white matter (WM) vessels with antibodies specific for collagens I, III and IV to test the hypothesis that GM vessels are immature. Brains were collected during post-mortem from prematurely born human neonates ranging in age from 17 weeks to 36 weeks postconception. All GM vessels were immunoreactive for collagen subtypes I, III and IV. Using digital image analysis, collagen IV immunoperoxidase-labelling was measured in vessels in GM, cortex and WM. Intensity values in GM and WM were normalized relative to cortical intensity within the same subject. At week 17 of gestation, GM vessels exhibited a higher concentration of collagen IV than did WM or cortical vessels. Regression analysis demonstrated that collagen intensity in GM was greater than that in cortex and WM at all stages. We conclude that GM vessels in even the youngest, prematurely born, viable neonates do not exhibit evidence of structural immaturity. The high incidence of GM haemorrhage in premature neonates may be related to factors other than a deficiency in accumulated collagen.     

Development of integrins in the vasculature of germinal matrix,cerebral cortex,and white matter of fetuses and premature infants

Germinal matrix (GM) vasculature is selectively vulnerable to hemorrhage in premature infants during the first 48 hr of life. This is attributed to rapid angiogenesis of this brain region, resulting in formation of nascent vessels that show a paucity of pericytes and immaturity of extracellular matrix. Integrins are key regulators of angiogenesis and contribute to stabilization of cerebral vasculature by providing endothelial– and astrocyte–matrix adhesion. Therefore, we asked whether GM exhibited a distinct regional pattern of integrin expression that was dissimilar from that of the cerebral cortex and white matter in human fetuses and premature infants. To this end, we measured protein and gene expression of integrins in the GM, cortex, and white matter of human fetuses (15–22 weeks), premature infants (23–35 weeks), and mature infants (36–40 weeks). We found that protein levels of α5β1 integrin were greater in the GM than in the cortex or white matter by 1.6‐fold for both fetuses and premature infants. α5β1 integrin mRNA expression was higher in the GM than in the cortex or white matter by 2‐fold for fetuses but not for premature infants. αVβ3, αVβ5, αVβ8, and α4β1 integrin expression were comparable among GM, cortex, and white matter in fetuses and premature infants. Because α5β1 integrin is a central regulator of angiogenesis, its elevation in the GM of fetuses and premature infants indicates that this might be a key activator of endothelial proliferation in this brain region. We speculate that selective α5β1 integrin inhibition might suppress angiogenesis in the GM and thus prevent brain hemorrhage in premature infants. © 2009 Wiley‐Liss, Inc.     

Developmental change in type VI collagen in human cerebral vessels.

Vascular development in the human brain was studied by immunohistochemistry using an anti-type VI collagen antibody. Positive vessels were evident from an early gestational age in the meninges, from 21 weeks gestation in the basal ganglia and deep white matter, and from 38 weeks gestation in the cerebral cortex and superficial white matter; however, type VI collagen never appeared in the subependymal germinal layer. The absent or scarce type VI collagen in the subependymal germinal layer may be one of the important factors of subependymal/intraventricular/periventricular hemorrhage in premature neonates. The earlier appearance of positive vessels in the deep white matter than in the cortex and superficial white matter suggests that the medullary vein develops earlier than the cortical and subcortical veins and arteries. These characteristics of the developing vascular structure may be one cause of perinatal brain damage.     

Temporal expression pattern of cerebrovascular endothelial cell alkaline phosphatase during human gestation

In premature human neonates, immaturity of cerebral vessels can contribute to clinical problems such as germinal matrix hemorrhage and white matter damage. Afferent cerebral vessels in the brain of term babies express alkaline phosphatase (AP), an ectoenzyme located on the surface of endothelial cells. Using AP enzyme histochemistry we have examined the cerebrovasculature of premature live-born human neonates to determine when cerebral afferent vessels begin to express AP. Brains were collected at autopsy and processed for histological examination. AP-stained vessel density in the periventricular white matter was quantified using digital imaging and automated morphometry. Babies born prior to 28 wk gestation display few AP-positive vessels in the periventricular white matter, whereas, babies born after 28 wk gestation exhibit an AP-positive vascular pattern that resembles the adult pattern. In contrast, immunostaining for collagen revealed an extensive vascular network in both early and late gestation infants. Our measurements indicate that neonates born prior to 28 wk gestation are characterized by immature cerebral white matter afferent vessels and raise the possibility that the immaturity compromises vascular function.     

Morphometry of blood vessels in the cortex and germinal plate of premature neonates

Germinal plate hemorrhage with intraventricular rupture has been a leading cause of morbidity and mortality among premature neonates. Because germinal plate hemorrhage decreases with increasing gestational age, germinal plate vessel size and/or structure may be an important etiologic factor. Electron micrographs of the cortical and germinal plate blood vessels from 13 premature infants ranging in age from 19-37 weeks gestation were studied; vessel, lumena, and endothelial areas were measured using a digitizing tablet. In infants between 25-32 weeks gestation, the germinal plate vessel and lumenal areas were significantly greater than the areas of cortical vessel and lumena. This finding suggests that one of the factors involved in germinal plate hemorrhage is described by LaPlace's law which states that the larger the vessel diameter, the greater the pressure on the vessel wall.     

Beagle pup germinal matrix maturation studies

Intraventricular hemorrhage, or hemorrhage into the germinal matrix tissues of the developing brain, remains a common problem of preterm infants. The "risk period" for this insult is the first 3-4 postnatal days. We hypothesized that this risk period for hemorrhage is related to rapid perinatal maturation of the germinal matrix vasculature and employed the newborn beagle pup model for the study of this maturation. Newborn beagle pups (n = 30) were anesthetized and systemically perfused with buffered formalin; the brains were removed and prepared for immunohistochemical study. Sections stained with Bandeiraea lectin demonstrated that there was no difference in germinal matrix vessel density between postnatal days 1 and 4. Germinal matrix sections were also stained for antibodies to alpha-smooth muscle actin, collagen IV, collagen V, desmin, factor VIII-related antigen, fibronectin, glial fibrillary acidic protein, laminin, transferrin, and vimentin. Vasculature staining by alpha-smooth muscle actin was not noted until postnatal day 10, and differential staining was detected for antibodies to laminin and collagen V. Quantification of staining intensity by confocal microscopy demonstrated a significant increase in both extracellular matrix components at postnatal day 4 compared with day 1 (p less than 0.05 for both). These basement membrane proteins may add sufficient structural integrity to germinal matrix vessels to prevent capillary rupture and thus intraventricular hemorrhage.     

A comprehensive review of therapeutic targets that induce microglia/macrophage-mediated hematoma resolution after germinal matrix hemorrhage

Currently, there is no effective treatment for germinal matrix hemorrhage and intraventricular hemorrhage (GMH-IVH), a common and often fatal stroke subtype in premature infants. Secondary brain injury after GMH-IVH is known to involve blood clots that contribute to inflammation and neurological deficits. Furthermore, the subsequent blood clots disrupt normal cerebrospinal fluid circulation and absorption after GMH-IVH, contributing to posthemorrhagic hydrocephalus (PHH). Clinically, GMH-IVH severity is graded on a I to IV scale: Grade I is confined to the germinal matrix, grade II includes intraventricular hemorrhage, grade III includes intraventricular hemorrhage with extension into dilated ventricles, and grade IV includes intraventricular hemorrhage with extension into dilated ventricles as well as parenchymal hemorrhaging. GMH-IVH hematoma volume is the best prognostic indicator, where patients with higher grades have worsened outcomes. Various preclinical studies have shown that rapid hematoma resolution quickly ameliorates inflammation and improves neurological outcomes. Current experimental evidence identifies alternatively activated microglia as playing a pivotal role in hematoma clearance. In this review, we discuss the pathophysiology of GMH-IVH in the development of PHH, microglia/macrophage's role in the neonatal CNS, and established/potential therapeutic targets that enhance M2 microglia/macrophage phagocytosis of blood clots after GMH-IVH.     

Neuropathologic substrate of cerebral palsy

Animal models have assisted in understanding the mechanisms of brain injury underlying cerebral palsy. Nevertheless, no such models replicate every aspect of the human disease. This review summarizes the classic and more recent studies of the neuropathology of human perinatal brain injury most commonly associated with cerebral palsy, for use by researchers and clinicians alike who need to analyze published animal models with respect to their fidelity to the human disorder. The neuropathology underlying cerebral palsy includes white-matter injury, known as periventricular leukomalacia, as well as germinal matrix hemorrhage with intraventricular extension, and injury to the cortex, basal ganglia, and thalamus. Each has distinctive features while sharing some risk factors, such as prematurity and/or hypoxia-ischemia in the perinatal period. Periventricular leukomalacia consists of diffuse injury of deep cerebral white matter, with or without focal necrosis. Recent work directly in human postmortem tissue has focused on the role of free radical injury, cytokine toxicity (especially in light of the epidemiologic association of periventricular leukomalacia with maternofetal infection), and excitotoxicity in the development of periventricular leukomalacia. Premyelinating oligodendrocytes, which predominate in periventricular regions during the window of vulnerability to periventricular leukomalacia (24-34 postconceptional weeks), are the targets of free radical injury, as determined by immunocytochemical markers of lipid peroxidation and protein nitration. This maturational susceptibility can be attributed in part to a relative deficiency of superoxide dismutases in developing white matter. Microglia, which respond to cytokines and to bacterial products such as lipopolysaccharide via Toll-like receptors, are increased in periventricular leukomalacia white matter and can contribute to cellular damage. Indeed, several cytokines, including tumor necrosis factor-a and interleukins 2 and 6, as well as interferon-g, have been demonstrated in periventricular leukomalacia. Preliminary work suggests a role for glutamate receptors and glutamate transporters in periventricular leukomalacia based on expression in human developing oligodendrocytes. Germinal matrix hemorrhage, with or without intraventricular hemorrhage, occurs in premature infants and can coexist with periventricular leukomalacia. Studies in human germinal matrix tissue have focused on maturation-based vascular factors, such as morphometry and expression of molecules related to the structure of the blood-brain barrier. Gray-matter injury, seen more commonly in term infants, includes cortical infarcts and status marmoratus. Subtle cortical injury overlying periventricular leukomalacia is the subject of current interest as a possible substrate for the cognitive difficulties seen in patients with cerebral palsy. In summary, it is hoped that work in human tissue, in conjunction with experimental animal models, will lead to eventual therapeutic or preventive strategies for the perinatal brain injury underlying cerebral palsy.     

Morphometric analysis of the human infant brain: effects of intraventricular hemorrhage and periventricular leukomalacia

Absolute and relative growth of cerebral structures was assessed morphometrically in 34 neurologically normal live-born infants surviving up to 1 year of age, 20 infants with fatal intraventricular hemorrhage, and 4 with periventricular leukomalacia. In the normal brain, cerebral cortex grew 25% faster than white matter throughout infancy. Thus, to achieve the normal adult size and 1:1 ratio of cortex to white matter, white matter must continue to grow for several years after cessation of cortical growth. Brains with intraventricular hemorrhage or periventricular leukomalacia manifested accelerated growth of gray-matter structures compared with controls. In addition, brains with periventricular leukomalacia had markedly depressed rates of white-matter growth compared with controls of similar age, whereas brains with intraventricular hemorrhage had disproportionate hydrocephalus. The findings suggest that (1) gray-matter growth is accelerated in premature infants with intraventricular hemorrhage or periventricular leukomalacia, perhaps reflecting perinatal stress; (2) periventricular leukomalacia is associated with impaired growth of white matter during a critical period; and (3) sequential quantitative assessment of ventricular size following intraventricular hemorrhage may assist in early recognition of progressive posthemorrhagic hydrocephalus.     

Immunohistochemical study of the vasculature in the developing brain

In this study, the developmental proliferation of human brain vessels, from the fetal to the adult stage, was analyzed by immunohistochemical methods using antitype IV collagen, antilaminin, and antifibronectin antibodies. Examination of the frontal lobe indicates that these antibodies bind to the vessels, both arteries and veins. During cortical angiogenesis, the density and diameter of vessels increase rapidly from about 26 weeks gestation and peak at 35 weeks; after 35 weeks, the density and diameter of vessels are the same as those in adult brain. The white matter demonstrates no major changes in vessel density, although the pattern of the changes in vessel diameter resembles that of the cortex. Small immunopositive spots suggesting neovascularization reveal the same developmental tendency as the density of vessels in the cortex and white matter; therefore, it appears that neovascularization in the fetal brain during development is more rapid than cortical expansion and is equal to the growth of white matter. Neovascularization may be closely related to normal brain development and may play an undefined role in perinatal cerebrovascular insults.     

Intraventricular hemorrhage in the premature infant--current concepts. Part I

Although the incidence of periventricular-intraventricular hemorrhage (IVH) has decreased in recent years, the increasing survival rates for the smallest premature infants indicate that the lesion will continue to be a major problem in neonatal intensive care facilities. The neuropathology is characterized by an elemental lesion, bleeding into the subependymal germinal matrix, with subsequent rupture into the lateral ventricle. Important neuropathological consequences are germinal matrix destruction, posthemorrhagic hydrocephalus, and periventricular hemorrhagic infarction. The last of these appears to be a venous infarction and is a critical determinant of neurological outcome. Neuropathological accompaniments, not caused by the IVH, include periventricular leukomalacia and pontine neuronal necrosis. The pathogenesis of IVHs is related to intravascular, vascular, and extravascular factors. Intravascular factors involve primarily control of blood flow and pressure in the microcirculation of the germinal matrix. Particular pathogenetic importance can be attached to fluctuations in cerebral blood flow, abrupt increases in flow, decreases in flow with injury to matrix vessels, increases in cerebral venous pressure, and, in selected infants, disturbances of platelet function and coagulation. Vascular factors relate to the microcirculation of the matrix, the site of the initial bleeding. A maturation-dependent alteration in vascular integrity and a vulnerability of matrix vessels to ischemic injury appear important. Extravascular factors include those relevant to mesenchymal and glial support for matrix vessels and to local fibrinolytic activity in the germinal matrix. The latter may be a manifestation of the proteolytic activity now recognized to be of general importance in developmental remodeling of the mammalian central nervous system.     

Early ontogenic disturbances in cell migration in mentally disabled adult

Disturbances in cell migration are heterogenic disorders of brain development commonly associated with epilepsy and mental retardation. We report a 45-year-old oligophrenic man with defect of lower limbs and family history of mental retardation who died because of brainstem hemorrhage. At post-mortem and histopathological examination, complex brain malformation characterized by bilateral periventricular heterotopia, cortical dysgenesia, partial agenesia of corpus callosum and thin-walled blood vessels was found. Immunohistochemical examination revealed the presence of fibronectin, collagen IV and laminin in wall of pathological vessels. Cyclooxygenase-2 (COX-2) expression in neurons within heterotopias and dysgenic cortex was negative. It may indicate their maturity and indirectly, normal activity of postsynaptic NMDA receptors that explains absence of epileptic attacks in our patient. The presence of COX-2-negative neurons and compounds of basal lamina in fetal-like vessels within heterotopias and dysgenic cortex, suggests that these probably initially immature structures became mature during 45 years of patient's life.     

Regional variability of blood flow and glucose utilization within the subependymal germinal matrix

Regional cerebral blood flow (RCBF) and local cerebral glucose utilization (LCGU) were studied in normal, awake newborn beagle puppies using quantitative autoradiography. A highly significant linear relationship between RCBF and LCGU was present for all brain structures. Special attention was directed to the subependymal germinal matrix (GM), the site of origin of periventricular hemorrhage in the premature human. A consistent variability in both RCBF and LCGU was found within GM. Both RCBF and LCGU were higher in rostral than posterior GM, and in peripheral than internal GM. The close coupling between RCBF and LCGU within GM suggests that GM blood flow is influenced by local energy needs. The quantitative relationship between RCBF and LCGU was similar within GM to that in cerebral white and gray matter, suggesting that the puppy GM predominantly utilizes glucose as fuel for energy production.     

Positive rolandic sharp waves in the EEG of the premature infant

Ninety-seven EEGs from 30 premature infants found to have multifocal white matter necrosis on ultrasound (US) or autopsy were reviewed retrospectively. Twenty infants had intraparenchymal echodensities on US that developed into cystic lesions, a finding consistent with periventricular leukomalacia; 8 had intraparenchymal hemorrhages; and 2 had white matter necrosis at autopsy. Four of these infants had no intraventricular hemorrhage. Positive sharp waves in the central (rolandic) regions (PRS) were identified in 22 of these 30 infants (73%) and in 0 of 30 age-matched controls (p less than 0.001). The presence of PRS on the EEG of the premature infant has a high correlation with white matter necrosis rather than with intraventricular hemorrhage. In all cases, this EEG pattern was present prior to the development of cavitations when echodensities were present on US.     

Effect of hyperoxic resuscitation on propensity of germinal matrix haemorrhage and cerebral injury

C. O. Chua, G. Vinukonda, F. Hu, N. Labinskyy, M. T. Zia, J. Pinto, A. Csiszar, Z. Ungvari and P. Ballabh (2010) Neuropathology and Applied Neurobiology 36, 448–458
Effect of hyperoxic resuscitation on propensity of germinal matrix haemorrhage and cerebral injury Aims: Intraventricular haemorrhage (IVH) and cerebral injury are major neurological disorders of premature infants. The effect of hyperoxic resuscitation on the occurrence of IVH and cerebral injury is elusive. Therefore, we asked whether hyperoxia during neonatal resuscitation increased the incidence and severity of IVH and cerebral injury in premature newborns. Methods: Premature rabbit pups, delivered by C‐section, were sequentially assigned to receive 100%, 40% or 21% oxygen for 15 or 60 min at birth. The pups were treated with intraperitoneal glycerol at 24‐h postnatal age to determine the incidence and severity of glycerol‐induced IVH. Vascular endothelial growth factor and angiopoietin‐2 genes and protein expression, endothelial proliferation as well as free radical levels and antioxidants were assessed in the germinal matrix, white matter and cortex of pups exposed to 100% oxygen and to 21% oxygen. Results: Exposure with 100% oxygen for 1 h did not adversely exacerbate the incidence of glycerol‐induced IVH in premature rabbit pups. Compared with room air, 100% oxygen enhanced mRNA expression of both vascular endothelial growth factor and angiopoietin‐2 as well as reactive oxygen species levels in the germinal matrix. Hyperoxia did not affect endothelial proliferation, apoptosis or neuronal degeneration in the forebrain. Conclusion: Our data suggest that 100% oxygen exposure for 1 h does not increase the risk of IVH or cerebral injury in premature rabbit pups. Although extrapolating rabbit neural developmental data into humans has obvious limitations, we speculate that hyperoxia of short duration at birth in premature infants may not result in major neurological adverse effects.     

Germinal matrix hemorrhage in Zellweger syndrome

A term male newborn was noted to have severe diffuse hypotonia, hyporeflexia, hepatosplenomegaly, and characteristic abnormal facies of Zellweger syndrome, the diagnosis of which was confirmed by identification of 2 mutations including Nt2098insT, a frameshift with premature stop codon in exon 13, as well as a novel second mutation at Nt3038G→A (Arg1013His) on skin fibroblast testing. His brain magnetic resonance imaging (MRI) demonstrated bilateral germinolytic cysts with unilateral hemorrhagic transformation. Germinolytic cysts are one of the characteristic radiographic features of Zellweger syndrome, but germinal matrix hemorrhage has never been reported. Germinal matrix hemorrhage is common in premature infants, but found in only 4% of normal term infants. Germinal matrix hemorrhage was seen in a case of Zellweger syndrome with a novel mutation.     

Subependymal veins in premature neonates: implications for hemorrhage

The germinal matrix contains a concentrated network of blood vessels. The unusual structural qualities of these vessels are implicated as a factor underlying the high incidence of hemorrhage that occurs in the germinal matrix of prematurely born neonates. The present study is a histologic analysis of an postmortem examination series of brains collected from neonates born between 23 weeks gestation and term and is designed to determine if subependymal veins can be recognized in neonates born at the limits of viability, approximately 23 weeks gestation. Alkaline phosphatase histochemistry is used to differentiate cerebral afferent from efferent vessels. The results demonstrate that precursors of the subependymal veins can be recognized as early as the twenty-third gestational week. These veins increase progressively in diameter from 23 weeks to term, but the wall of the veins, which at early stages consists of endothelial cells only, does not thicken until after postconception week 36. Thus in all premature neonates, including the youngest capable of independent existence, the subependymal veins are present and appear vulnerable to rupture. These data support our suggestion that the structural immaturity of these veins in premature neonates is causally related to the high incidence of germinal matrix hemorrhage in these patients.     

Intraventricular hemorrhage and developmental outcomes at 24 months of age in extremely preterm infants

Whether intraventricular hemorrhage increases the risk of adverse developmental outcome among premature infants is controversial. Using brain ultrasound, we identified intraventricular hemorrhage and white matter abnormalities among 1064 infants born before 28 weeks' gestation. We identified adverse developmental outcomes at 24 months of age using a standardized neurologic examination and the Bayley Scales of Infant Development Mental and Motor Scales. In logistic regression models that adjusted for gestational age, sex, and public insurance, isolated intraventricular hemorrhage was associated with visual fixation difficulty but no other adverse outcome. Infants who had a white matter lesion unaccompanied by intraventricular hemorrhage were at increased risk of cerebral palsy, low Mental and Motor Scores, and visual and hearing impairments. Except when accompanied or followed by a white matter lesion, intraventricular hemorrhage is associated with no more than a modest increase (and possibly no increase) in the risk of adverse developmental outcome during infancy.     

Ventriculosubgaleal shunt procedure and its long-term outcomes in premature infants with post-hemorrhagic hydrocephalus

Objective  

It is well known that 10–15% of hydrocephalus cases at childhood and 40–50% in premature infants, occur following Germinal matrix hemorrhage (GMH). Such hemorrhages are reported to arise due to the rupture of germinal matrix (GM) vessels as a result of cerebral blod flow changes among infants with <1500 g birth weight and <32 weeks old. Intraventricular hemorrhage (IVH) associated with GMH leads to a disruption in the cerebrospinal fluid (CSF) and ventricular dilatation. Ventriculosubgaleal shunt (VSGS) is preferred in those hydrocephalus cases because it is a simple and rapid method, precludes the need for repetitive aspiration for evacuation of CSF, establishes a permanent decompression without causing electrolyte and nutritional losses, and aims to protect the cerebral development of newborns with GMH.