Microstructural Alterations and Oligodendrocyte Dysmaturation in White Matter After Cardiopulmonary Bypass in a Juvenile Porcine Model.

BACKGROUND: Newly developed white matter (WM) injury is common after cardiopulmonary bypass (CPB) in severe/complex congenital heart disease. Fractional anisotropy (FA) allows measurement of macroscopic organization of WM pathology but has rarely been applied after CPB. The aims of our animal study were to define CPB-induced FA alterations and to determine correlations between these changes and cellular events after congenital heart disease surgery. METHODS AND RESULTS: Normal porcine WM development was first assessed between 3 and 7 weeks of age: 3-week-old piglets were randomly assigned to 1 of 3 CPB-induced insults. FA was analyzed in 31 WM structures. WM oligodendrocytes, astrocytes, and microglia were assessed immunohistologically. Normal porcine WM development resembles human WM development in early infancy. We found region-specific WM vulnerability to insults associated with CPB. FA changes after CPB were also insult dependent. Within various WM areas, WM within the frontal cortex was susceptible, suggesting that FA in the frontal cortex should be a biomarker for WM injury after CPB. FA increases occur parallel to cellular processes of WM maturation during normal development; however, they are altered following surgery. CPB-induced oligodendrocyte dysmaturation, astrogliosis, and microglial expansion affect these changes. FA enabled capturing CPB-induced cellular events 4 weeks postoperatively. Regions most resilient to CPB-induced FA reduction were those that maintained mature oligodendrocytes. CONCLUSIONS: Reducing alterations of oligodendrocyte development in the frontal cortex can be both a metric and a goal to improve neurodevelopmental impairment in the congenital heart disease population. Studies using this model can provide important data needed to better interpret human imaging studies

Aprotinin, but not ε-aminocaproic acid and tranexamic acid, exerts neuroprotection against excitotoxic injury in an in vitro neuronal cell culture model

ObjectiveLack of availability of aprotinin has resulted in increased clinical use of the alternative antifibrinolytic agents, ε-aminocaproic acid (EACA) and tranexamic acid (TXA), which are known to be associated with an increased risk of seizures. In contrast, aprotinin has previously been demonstrated to be neuroprotective through suppression of excitotoxicity-mediated neuronal degeneration via the extracellular plasminogen/plasmin system. This study compares the effect of antifibrinolytic agents on neuronal and mixed glial/neuronal cell cultures.MethodsMixed cortical cultures containing neuronal and glial cells were prepared from fetal mice and plated on a layer of confluent astrocytes from postnatal pups. A primary neuronal culture was obtained from the same gestational stage and plated in multiwall vessels. Slowly triggered excitotoxicity was induced by 24-hour exposure to 12.5 mM N-methyl-d-aspartate (NMDA). Apoptotic neuronal cell death was induced by exposure of primary neural cultures to 24 hours of serum deprivation.ResultsCompared with NMDA alone, no significant changes in cell death were observed for any dose of TXA or EACA in mixed cultures. Conversely, a clinical dose of aprotinin significantly reduced cell death by −31% on average. Aprotinin reduced apoptotic neuronal cell death from 75% to 37.3%, and to 34.1% at concentrations of 100 and 200 kIU/mL, respectively, and significantly decreased neuronal nuclear damage. These concentrations of aprotinin significantly inhibited caspase 9 and 3/7 activations; 250 kIU/mL aprotinin exerted maximal protection on primary cortical neurons.ConclusionsIn contrast to aprotinin, EACA and TXA exert no protective effect against excitotoxic neuronal injury that can occur during cardiac surgery

Rodent brain slice model for the study of white matter injury

ObjectiveCerebral white matter (WM) injury is common after cardiac surgery in neonates and young infants who have brain immaturity and genetic abnormalities. To understand better the mechanisms associated with WM injury, we tested the adequacy of a novel ex vivo brain slice model, with a particular focus on how the maturational stage modulates the injury.MethodsTo replicate conditions of cardiopulmonary bypass, we transferred living brain slices to a closed chamber perfused by artificial cerebrospinal fluid under controlled temperature and oxygenation. Oxygen-glucose deprivation (OGD) simulated circulatory arrest. The effects of maturation were investigated in 7- and 21-day-old mice (P7, P21) that are equivalent in maturation stage to the human fetus and young adult.ResultsThere were no morphologic changes in axons after 60 minutes of OGD at 15°C in both P7 WM and P21 WM. Higher temperature and longer duration of OGD were associated with significantly greater WM axonal damage, suggesting that the model replicates the injury seen after hypothermic circulatory arrest. The axonal damage at P7 was significantly less than at P21, demonstrating that immature axons are more resistant than mature axons. Conversely, a significant increase in caspase3+ oligodendrocytes in P7 mice was identified relative to P21, indicating that oligodendrocytes in immature WM are more vulnerable than oligodendrocytes in mature WM.ConclusionsNeuroprotective strategies for immature WM may need to focus on reducing oligodendrocyte injury. The brain slice model will be helpful in understanding the effects of cardiac surgery on the immature brain and the brain with genetic abnormalities

Hypoxia Results in White Matter Immaturity in a Piglet Model of Congenital Heart Disease

Congenital heart disease (CHD) is the leading birth defect, affecting almost 1% of births each year. Full-term infants with CHD display subnormal brain development, underlying impairments in fine/gross motor skills, language, and memory. CHD infants have a high incidence of brain injury; partly due to insufficiencies in cerebral oxygen delivery in utero. Diffusion tensor imaging (DTI) studies have revealed that white matter (WM) immaturity is common in infants with CHD. Due to technical and ethical difficulties, the effects of CHD-induced brain injury on the cellular level remain elusive. To emulate insufficient cerebral oxygenation in CHD, we developed a porcine chronic hypoxia model and analyzed the microstructural and cellular effects of CHD on/in the corpus callosum (CC) with DTI and immunohistochemistry, respectively. Fixed porcine brains were imaged with a 3T-magnet at Johns Hopkins University. The cerebrum was isolated from DTI images using ROI Editor and fiber tracking was performed using DTI Studio. The primary antibodies used were PDGFR-α to label oligodendrocyte (OL) progenitors, CC1 to label mature OLs, Casp3 to label apoptotic cells, and Ki67 to label proliferating cells. To ensure an unbiased assessment, cell counts were performed using Stereology. DTI analysis demonstrated that hypoxia leads to a global reduction in the number and length of WM fiber tracts along with a decrease in fractional anisotropy-a metric of WM integrity and maturity. Immunohistochemical analyses revealed a 75% decrease in the density of apoptotic mature OLs and an 85% decrease in the density of proliferating OL progenitors in the CC following hypoxia (p\u3c0.05). Together, these findings indicate an OL lineage-specific vulnerability to hypoxic exposure where OL progenitors fail to generate new OLs at a rate necessary for normal brain development. Hence, therapies aimed at restoring the regenerative capacity of resident OL progenitors within the CC offer promising avenues to improving neurological outcomes in the growing CHD population

Prolonged white matter inflammation after cardiopulmonary bypass and circulatory arrest in a juvenile porcine model.

BACKGROUND: White matter (WM) injury is common after neonatal cardiopulmonary bypass (CPB). We have demonstrated that the inflammatory response to CPB is an important mechanism of WM injury. Microglia are brain-specific immune cells that respond to inflammation and can exacerbate injury. We hypothesized that microglia activation contributes to WM injury caused by CPB. METHODS: Juvenile piglets were randomly assigned to one of three CPB-induced brain insults (1: no-CPB, 2: full-flow CPB, 3: CPB/Circulatory-arrest). Neurobehavioral tests were performed. Animals were sacrificed 3-days or 4-weeks post-operatively. Microglia and proliferating cells were immunohistologically identified. Seven analyzed WM regions were further categorized into 3-fiber connections (1: Commissural, 2: Projection, 3: Association fibers). RESULTS: Microglia numbers significantly increased on day 3 after CPB/Circulatory-arrest, but not after full-flow CPB. Fiber categories did not affect these changes. On post-CPB week 4, proliferating cell number, blood leukocyte number and IL-6 levels, and neurological scores had normalized. However, both full-flow CPB and CPB/Circulatory-arrest displayed significant increases in the microglia number compared with Control. Thus brain-specific inflammation after CPB persists despite no changes in systemic biomarkers. Microglia number was significantly different among fiber categories, being highest in Association and lowest in Commissural connections. Thus there was a WM fiber-dependent microglia reaction to CPB. CONCLUSIONS: This study demonstrates prolonged microglia activation in WM after CPB. This brain-specific inflammatory response is systemically silent. It is connection fiber-dependent which may impact specific connectivity deficits observed after CPB. Controlling microglia activation after CPB is a potential therapeutic intervention to limit neurological deficits following CPB

White matter protection in congenital heart surgery

BACKGROUND: Neurodevelopmental delays in motor skills and white matter (WM) injury have been documented in congenital heart disease (CHD) and after pediatric cardiac surgery. The lack of a suitable animal model has hampered our understanding of the cellular mechanisms underlying WM injury in these patients. Our aim is to identify an optimal surgical strategy for WM protection to reduce neurological injury in CHD patients. METHODS AND RESULTS: We developed a porcine cardiopulmonary bypass (CPB) model, which displays area dependent WM maturation. In this model, WM injury was identified following CPB-induced ischemia-reperfusion injury. The degree of injury was inversely correlated with the maturation stage, indicating maturation-dependent vulnerability of WM. Within different oligodendrocyte (OL) developmental stages, we show selective vulnerability of O4(+) pre-OLs, while OL progenitor cells (OPCs) were resistant to insults. This indicates that immature WM is vulnerable to CPB-induced injury, but has an intrinsic potential for recovery mediated by endogenous OPCs. OPC number decreased with age, suggesting that earlier repair allows successful WM development. OPC proliferation was observed within a few days after CPB-induced ischemia-reperfusion injury; however by four weeks arrested OL maturation and delayed myelination were detected. Logistic model confirmed that maintaining higher oxygenation and reducing inflammation were effective in minimizing the risk of injury at immature stages of WM development. CONCLUSIONS: Primary repair in neonates and young infants potentially provides successful WM development in CHD patients. Cardiac surgery during this susceptible period should avoid ischemia-reperfusion injury and minimize inflammation to prevent long-term WM-related neurological impairment

Microstructural Alterations and Oligodendrocyte Dysmaturation in White Matter After Cardiopulmonary Bypass in a Juvenile Porcine Model

BACKGROUND: Newly developed white matter (WM) injury is common after cardiopulmonary bypass (CPB) in severe/complex congenital heart disease. Fractional anisotropy (FA) allows measurement of macroscopic organization of WM pathology but has rarely been applied after CPB. The aims of our animal study were to define CPB-induced FA alterations and to determine correlations between these changes and cellular events after congenital heart disease surgery. METHODS AND RESULTS: Normal porcine WM development was first assessed between 3 and 7 weeks of age: 3-week-old piglets were randomly assigned to 1 of 3 CPB-induced insults. FA was analyzed in 31 WM structures. WM oligodendrocytes, astrocytes, and microglia were assessed immunohistologically. Normal porcine WM development resembles human WM development in early infancy. We found region-specific WM vulnerability to insults associated with CPB. FA changes after CPB were also insult dependent. Within various WM areas, WM within the frontal cortex was susceptible, suggesting that FA in the frontal cortex should be a biomarker for WM injury after CPB. FA increases occur parallel to cellular processes of WM maturation during normal development; however, they are altered following surgery. CPB-induced oligodendrocyte dysmaturation, astrogliosis, and microglial expansion affect these changes. FA enabled capturing CPB-induced cellular events 4 weeks postoperatively. Regions most resilient to CPB-induced FA reduction were those that maintained mature oligodendrocytes. CONCLUSIONS: Reducing alterations of oligodendrocyte development in the frontal cortex can be both a metric and a goal to improve neurodevelopmental impairment in the congenital heart disease population. Studies using this model can provide important data needed to better interpret human imaging studies