Ludmila Korotcova

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 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 congenital heart disease patients. Methods and Results— We developed a porcine cardiopulmonary bypass model that displays area-dependent WM maturation. In this model, WM injury was identified after cardiopulmonary bypass–induced ischemia-reperfusion injury. The degree of injury was inversely correlated with the maturation stage, which indicates maturation-dependent vulnerability of WM. Within different oligodendrocyte developmental stages, we show selective vulnerability of O4+ preoligodendrocytes, whereas oligodendrocyte progenitor cells were resistant to insults. This indicates that immature WM is vulnerable to cardiopulmonary bypass–induced injury but has an intrinsic potential for recovery mediated by endogenous oligodendrocyte progenitor cells. Oligodendrocyte progenitor cell number decreased with age, which suggests that earlier repair allows successful WM development. Oligodendrocyte progenitor cell proliferation was observed within a few days after cardiopulmonary bypass–induced ischemia-reperfusion injury; however, by 4 weeks, arrested oligodendrocyte maturation and delayed myelination were detected. Logistic model confirmed that maintenance of higher oxygenation and reduction of 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 congenital heart disease patients. Cardiac surgery during this susceptible period should avoid ischemia-reperfusion injury and minimize inflammation to prevent long-term WM-related neurological impairment.

Effects of preoperative hypoxia on white matter injury associated with cardiopulmonary bypass in a rodent hypoxic and brain slice model.

Background:White matter (WM) injury is common after cardiopulmonary bypass or deep hypothermic circulatory arrest in neonates who have cerebral immaturity secondary to in utero hypoxia. The mechanism remains unknown. We investigated effects of preoperative hypoxia on deep hypothermic circulatory arrest–induced WM injury using a combined experimental paradigm in rodents.Methods:Mice were exposed to hypoxia (prehypoxia). Oxygen–glucose deprivation was performed under three temperatures to simulate brain conditions of deep hypothermic circulatory arrest including ischemia–reperfusion/reoxygenation under hypothermia.Results:WM injury in prenormoxia was identified after 35 °C-oxygen–glucose deprivation. In prehypoxia, injury was displayed in all groups. Among oligodendrocyte stages, the preoligodendrocyte was the most susceptible, while the oligodendrocyte progenitor was resistant to insult. When effects of prehypoxia were assessed, injury of mature oligodendrocytes and oligodendrocyte progenitors in prehypoxia significantly increased as compared with prenormoxia, indicating that mature oligodendrocytes and progenitors that had developed under hypoxia had greater vulnerability. Conversely, damage of oligodendrocyte progenitors in prehypoxia were not identified after 15 °C-oxygen–glucose deprivation, suggesting that susceptible oligodendrocytes exposed to hypoxia are protected by deep hypothermia.Conclusion:Developmental alterations due to hypoxia result in an increased WM susceptibility to injury. Promoting WM regeneration by oligodendrocyte progenitors after earlier surgery using deep hypothermia is the most promising approach for successful WM development in congenital heart disease patients.

Hypoxia diminishes the protective function of white-matter astrocytes in the developing brain.

OBJECTIVESnWhite-matter injury after surgery is common in neonates with cerebral immaturity secondary to in utero hypoxia. Astrocytes play a central role in brain protection; however, the reaction of astrocytes to hypothermic circulatory arrest (HCA) remains unknown. We investigated the role of astrocytes in white-matter injury after HCA and determined the effects of preoperative hypoxia on this role, using a novel mouse model.nnnMETHODSnMice were exposed to hypoxia from days 3 to 11, which is equivalent to the third trimester in humans (prehypoxia, n = 49). Brain slices were transferred to a chamber perfused by cerebrospinal fluid. Oxygen-glucose deprivation (OGD) was performed to simulate ischemia-reperfusion/reoxygenation resulting from circulatory arrest under hypothermia. Astrocyte reactions were compared with preoperative normoxia (prenormoxia; n = 45).nnnRESULTSnWe observed astrocyte activation after 25°C ischemia-reperfusion/reoxygenation in prenormoxia (P < .01). Astrocyte number after OGD correlated with caspase-3(+) cells (rho = .77, P = .001), confirming that astrogliosis is an important response after HCA. At 3 hours after OGD, astrocytes in prenormoxia had already proliferated and become activated (P < .05). Conversely, astrocytes that developed under hypoxia did not display these responses. At 20 hours after ischemia-reperfusion/reoxygenation, astrogliosis was not observed in prehypoxia, demonstrating that hypoxia altered the response of astrocytes to insult. In contrast to prenormoxia, caspase-3(+) cells in prehypoxia increased after ischemia reperfusion/reoxygenation, compared with control (P < .01). Caspase-3(+) cells were more common with prehypoxia than with prenormoxia (P < .001), suggesting that lack of astrogliosis permits increased white-matter injury.nnnCONCLUSIONSnPreoperative hypoxia alters the neuroprotective function of astrocytes. Restoring this function before surgery may be a therapeutic option to reduce postoperative white-matter injury in the immature brain.

Rodent brain slice model for the study of white matter injury

OBJECTIVEnCerebral 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 exxa0vivo brain slice model, with a particular focus on how the maturational stage modulates the injury.nnnMETHODSnTo 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.nnnRESULTSnThere 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.nnnCONCLUSIONSnNeuroprotective 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.

Abnormal neurogenesis and cortical growth in congenital heart disease

Congenital heart disease depletes SVZ neural stem/progenitor cell pools critical for normal cortical growth. Getting to the heart of the matter in brain development Congenital heart disease (CHD), the most common birth defect in newborns, can be associated with developmental delays. Although reduced blood flow, genetic factors, and brain injury are thought to contribute, the cellular mechanisms underlying abnormal brain development due to CHD are unclear. Morton et al. used a piglet model of neonatal hypoxia to study the relationship between neural stem/progenitor cells and cortical development. Chronic hypoxia reduced the number of stem/progenitor cells within the subventricular zone in piglet brains, which limited the number of interneurons and cortical growth. These findings were also seen in brain tissue from human infants with CHD. Long-term neurological deficits due to immature cortical development are emerging as a major challenge in congenital heart disease (CHD). However, cellular mechanisms underlying dysregulation of perinatal corticogenesis in CHD remain elusive. The subventricular zone (SVZ) represents the largest postnatal niche of neural stem/progenitor cells (NSPCs). We show that the piglet SVZ resembles its human counterpart and displays robust postnatal neurogenesis. We present evidence that SVZ NSPCs migrate to the frontal cortex and differentiate into interneurons in a region-specific manner. Hypoxic exposure of the gyrencephalic piglet brain recapitulates CHD-induced impaired cortical development. Hypoxia reduces proliferation and neurogenesis in the SVZ, which is accompanied by reduced cortical growth. We demonstrate a similar reduction in neuroblasts within the SVZ of human infants born with CHD. Our findings demonstrate that SVZ NSPCs contribute to perinatal corticogenesis and suggest that restoration of SVZ NSPCs’ neurogenic potential is a candidate therapeutic target for improving cortical growth in CHD.

Prolonged White Matter Inflammation After Cardiopulmonary Bypass and Circulatory Arrest in a Juvenile Porcine Model

BACKGROUNDnWhite 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.nnnMETHODSnJuvenile piglets were randomly assigned to 1 of 3 CPB-induced brain insults (1, no-CPB; 2, full-flow CPB; 3, CPB and circulatory arrest). Neurobehavioral tests were performed. Animals were sacrificed 3 days or 4 weeks postoperatively. 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).nnnRESULTSnMicroglia numbers significantly increased on day 3 after CPB and 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, interleukin (IL)-6 levels, and neurologic scores had normalized. However, both full-flow CPB and CPB and 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.nnnCONCLUSIONSnThis 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 neurologic deficits after CPB.

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

OBJECTIVEnLack 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.nnnMETHODSnMixed 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.nnnRESULTSnCompared 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.nnnCONCLUSIONSnIn contrast to aprotinin, EACA and TXA exert no protective effect against excitotoxic neuronal injury that can occur during cardiac surgery.

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.

Prenatal/preoperative hypoxia diminishes the protective function of white matter astrocytes

s / Int. J. Devl Neuroscience 47 (2015) 1–131 45 brain seems to be correlated with neural injuries. We examined the behavioural improvement related to spatial memory through prevention of SD wave propagation in the brain, using excitatory neurotransmitter antagonist during early stage of growth. For this purpose KCl-induced rat used as a model of SD and AMPA-type glutamate receptors antagonist, DNQX, was selected for evaluating possible therapeutic effect. In our study, we administrated DNQX in an optimized dose (1 mg/kg) intraperitoneally after SD induction and SD was induced by 2 M KCL in juvenile Wistar rats (60–80 g). At the same time recording of SD wave were done to prove SD initiation and propagation, then DNXQ solution was applied through intra-peritoneal injection and their behaviour related to memory was studied by T-Maze test. Assessing the T-maze results, we observed that induction of SD in juvenile rats can cause memory impairment after four weeks in two-month-old rats. On the other hand, blockade of AMPA receptor with DNQX have not the ability to reduce the rate of memory impairment caused by SD. Our investigation showed that the efficacy of DNQX, as an AMPA receptor antagonist, is not correlated with memory improvement after impairment following SD, during early stage of growth. http://dx.doi.org/10.1016/j.ijdevneu.2015.04.126