Krishna Dummula

Consequences of Intraventricular Hemorrhage in a Rabbit Pup Model

Background and Purpose— Intraventricular hemorrhage (IVH) is a common complication of prematurity that results in neurological sequelae, including cerebral palsy, posthemorrhagic hydrocephalus, and cognitive deficits. Despite this, there is no standardized animal model exhibiting neurological consequences of IVH in prematurely delivered animals. We asked whether induction of moderate-to-severe IVH in premature rabbit pups would produce long-term sequelae of cerebral palsy, posthemorrhagic hydrocephalus, reduced myelination, and gliosis. Methods— The premature rabbit pups, delivered by cesarean section, were treated with intraperitoneal glycerol at 2 hours postnatal age to induce IVH. The development of IVH was diagnosed by head ultrasound at 24 hours of age. Neurobehavioral, histological, and ultrastructural evaluation and diffusion tensor imaging studies were performed at 2 weeks of age. Results— Although 25% of pups with IVH (IVH pups) developed motor impairment with hypertonia and 42% developed posthemorrhagic ventriculomegaly, pups without IVH (non-IVH) were unremarkable. Immunolabeling revealed reduced myelination in the white matter of IVH pups compared with saline- and glycerol-treated non-IVH controls. Reduced myelination was confirmed by Western blot analysis. There was evidence of gliosis in IVH pups. Ultrastructural studies in IVH pups showed that myelinated and unmyelinated fibers were relatively preserved except for focal axonal injury. Diffusion tensor imaging showed reduction in fractional anisotropy and white matter volume confirming white matter injury in IVH pups. Conclusion— The rabbit pups with IVH displayed posthemorrhagic ventriculomegaly, gliosis, reduced myelination, and motor deficits, like humans. The study highlights an instructive animal model of the neurological consequences of IVH, which can be used to evaluate strategies in the prevention and treatment of posthemorrhagic complications.

Bone Morphogenetic Protein Inhibition Promotes Neurological Recovery after Intraventricular Hemorrhage

Intraventricular hemorrhage (IVH) results in neural cell death and white matter injury in premature infants. No therapeutic strategy is currently available against this disorder. Bone morphogenetic protein (BMP) signaling suppresses oligodendrocyte development through basic-helix-loop-helix (bHLH) transcription factors and promotes astrocytosis. Therefore, we hypothesized that IVH in premature newborns initiates degeneration and maturation arrest of oligodendrocyte lineage and that BMP inhibition alleviates hypomyelination, gliosis, and motor impairment in the survivors of IVH. To test the hypotheses, a rabbit model of IVH was used in which premature rabbit pups (E29) are treated with intraperitoneal glycerol at 2 h of age to induce IVH; and the pups with IVH exhibit hypomyelination and gliosis at 2 weeks of postnatal age. Maturation of oligodendrocyte lineage was evaluated by specific markers, and the expression of bHLH transcription factors was assessed. BMP levels were measured in both premature rabbit pups and autopsy materials from premature infants. Recombinant human noggin was used to suppress BMP action; and neurobehavioral performance, myelination and gliosis were assessed in noggin-treated pups compared with untreated controls. We found that IVH resulted in apoptosis and reduced proliferation of oligodendrocyte progenitors, as well as arrested maturation of preoligodendrocytes in rabbits. BMP4 levels were significantly elevated in both rabbit pups and human premature infants with IVH compared with controls. Importantly, BMP inhibition by recombinant human noggin restored the levels of phospho-Smad1/5/8, Olig2 transcription factor, oligodendrocyte maturation, myelination, astrocyte morphology, and motor function in premature pups with IVH. Hence, BMP inhibition might enhance neurological recovery in premature infants with IVH.

Effect of Prenatal Glucocorticoids on Cerebral Vasculature of the Developing Brain

Background and Purpose— Prenatal glucocorticoids prevent germinal matrix hemorrhage in premature infants. The underlying mechanism, however, is elusive. Germinal matrix is enriched with angiogenic vessels exhibiting paucity of pericytes and glial fibrillary acidic protein-positive astrocyte end feet. Therefore, we asked whether glucocorticoid treatment would suppress angiogenesis and enhance periendothelial coverage by pericytes and glial fibrillary acidic protein-positive end feet in the germinal matrix microvasculature. Methods— We treated pregnant rabbits with intramuscular betamethasone and delivered pups prematurely by cesarean section at E29 (term=32 days). Endothelial turnover, vascular density, pericyte coverage, glial fibrillary acidic protein-positive end feet, cell death, and growth factors orchestrating angiogenesis, including vascular endothelial growth factor, angiopoietins, transforming growth factor-&bgr;, and platelet-derived growth factor-B, were compared between betamethasone-treated and untreated pups. Similar comparisons were done between autopsy materials from premature infants exposed and unexposed to prenatal glucocorticoids. Results— Antenatal glucocorticoid treatment reduced endothelial proliferation, vascular density, and vascular endothelial growth factor expression in the germinal matrix of both rabbits and humans. The pericyte coverage was greater in glucocorticoid-treated rabbit pups and human infants than in controls, but not the glial fibrillary acidic protein-positive end feet coverage. Transforming growth factor-&bgr;, but not angiopoietins and platelet-derived growth factor-B, were elevated in glucocorticoid-treated rabbit pups compared with controls. Betamethasone treatment induced apoptosis, neuronal degeneration, and gliosis in rabbit pups. However, there was no evidence of increased cell death in glucocorticoid-exposed human infants. Conclusions— Prenatal glucocorticoid suppresses vascular endothelial growth factor and elevates transforming growth factor-&bgr; levels, which results in angiogenic inhibition, trimming of neovasculature, and enhanced pericyte coverage. These changes contribute to stabilizing the germinal matrix vasculature, thereby reducing its propensity to hemorrhage. Prenatal glucocorticoid exposure does not induce neural cell death in humans, unlike rabbits.

Neuroprotection in a rabbit model of intraventricular haemorrhage by cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-alpha inhibition

Intraventricular haemorrhage is a major complication of prematurity that results in neurological dysfunctions, including cerebral palsy and cognitive deficits. No therapeutic options are currently available to limit the catastrophic brain damage initiated by the development of intraventricular haemorrhage. As intraventricular haemorrhage leads to an inflammatory response, we asked whether cyclooxygenase-2, its derivative prostaglandin E2, prostanoid receptors and pro-inflammatory cytokines were elevated in intraventricular haemorrhage; whether their suppression would confer neuroprotection; and determined how cyclooxygenase-2 and cytokines were mechanistically-linked. To this end, we used our rabbit model of intraventricular haemorrhage where premature pups, delivered by Caesarian section, were treated with intraperitoneal glycerol at 2 h of age to induce haemorrhage. Intraventricular haemorrhage was diagnosed by head ultrasound at 6 h of age. The pups with intraventricular haemorrhage were treated with inhibitors of cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-α; and cell-infiltration, cell-death and gliosis were compared between treated-pups and vehicle-treated controls during the first 3 days of life. Neurobehavioural performance, myelination and gliosis were assessed in pups treated with cyclooxygenase-2 inhibitor compared to controls at Day 14. We found that both protein and messenger RNA expression of cyclooxygenase-2, prostaglandin E2, prostanoid receptor-1, tumour necrosis factor-α and interleukin-1β were consistently higher in the forebrain of pups with intraventricular haemorrhage relative to pups without intraventricular haemorrhage. However, cyclooxygenase-1 and prostanoid receptor 2-4 levels were comparable in pups with and without intraventricular haemorrhage. Cyclooxygenase-2, prostanoid receptor-1 or tumour necrosis factor-α inhibition reduced inflammatory cell infiltration, apoptosis, neuronal degeneration and gliosis around the ventricles of pups with intraventricular haemorrhage. Importantly, cyclooxygenase-2 inhibition alleviated neurological impairment, improved myelination and reduced gliosis at 2 weeks of age. Cyclooxygenase-2 or prostanoid receptor-1 inhibition reduced tumour necrosis factor-α level, but not interleukin-1β. Conversely, tumour necrosis factor-α antagonism did not affect cyclooxygenase-2 expression. Hence, prostanoid receptor-1 and tumour necrosis factor-α are downstream to cyclooxygenase-2 in the inflammatory cascade induced by intraventricular haemorrhage, and cyclooxygenase-2-inhibition or suppression of downstream molecules--prostanoid receptor-1 or tumour necrosis factor-α--might be a viable neuroprotective strategy for minimizing brain damage in premature infants with intraventricular haemorrhage.

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.

Antenatal thrombosis of torcular herophili presenting with anemia, consumption coagulopathy and high-output cardiac failure in a preterm infant.

Dural sinus malformation occasionally results in the development of giant venous lakes involving torcular herophili. Such dilatation can result in the formation of a massive venous thrombus leading to ventriculomegaly/hydrocephalus. Although, majority of patients have an unremarkable clinical course owing to spontaneous resolution of the thrombus, significant morbidity and mortality has been documented. We report the first case of torcular thrombosis in a preterm infant who survived severe anemia, consumption coagulopathy and high-output cardiac failure during the neonatal period and had a normal neurological outcome.

Misinterpretation of negative pulse oximetry screening as absence of critical congenital heart disease.

We read the case report by Harden et al. [2] entitled False-Negative Pulse Oximetry Screening for Critical CongenitalHeart Disease: The Case for Parent Education with greatinterest. This case underscores the importance of inter-preting screening test results with caution.Sincetheendorsementofpulseoximetryscreening(POS)for critical congenital heart disease (CCHD) by the Ameri-can AcademyofPediatrics(AAP)[3],manyhospitals acrossthe country, including ours, have proactively implementedthescreen.WeapplaudtheAAPfortheinitiativeandwishtohighlight through this letter some of the practical concernsarising after implementation of these recommendations.The AAP did not provide any standardized documenta-tion guidelines for incorporating the screening results in thedischarge summary to date. Nonstandard statements such as‘‘CCHD screen-negative’’ in the nursery discharge sum-maries may give rise to a false sense of reassurance to theunfamiliar medical practitioner and the parents that the childhas no possibility of critical congenital heart disease. Thepulse oximetry screen holds a very high specificity for rulingout certain CCHDs, as demonstrated in the Swedish [1] andGerman [4] studies. Clearly, other conditions are associatedwith significant morbidity and mortality that need ongoingsurveillance. Considering all these, we are concerned thatthere is room for overlooking serious congenital heart con-ditions if clinicians and parents are not adequately educatedconcerning the limitations of the screening protocol.We enthusiastically support the AAP’s endorsement of pulseoximetry screening for early detection of CCHD but urge theAcademy to mandate that nurseries document the cardiac con-ditions specifically ruled out by virtue of a negative screen onevery discharge summary. We urge the Academy also to con-sider expanding the algorithm to include comprehensive guide-linesforongoingsurveillanceforcongenitalheartdisease(CHD)during well-child visits of screen-negative children.The AAP’s recommendation calling for its members towork in concert with the local and regional chapters andrespective policy makers in the development of the edu-cational materials and training [3] is probably the best wayto ensure that no child with CHD is missed.