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Germinal matrix hemorrhage (GMH) is a common complication in preterm infants and is associated with high risk of adverse neurodevelopmental outcomes. We used a rat GMH model and performed RNA sequencing to investigate the signaling pathways and biological processes following hemorrhage. GMH induced brain injury characterized by early hematoma and subsequent tissue loss. At 6 hours after GMH, gene expression indicated an increase in mitochondrial activity such as ATP metabolism and oxidative phosphorylation along with upregulation of cytoprotective pathways and heme metabolism. At 24 hours after GMH, the expression pattern suggested an increase in cell cycle progression and downregulation of neurodevelopmental-related pathways. At 72 hours after GMH, there was an increase in genes related to inflammation and an upregulation of ferroptosis. Hemoglobin components and genes related to heme metabolism and ferroptosis such as Hmox1, Alox15, and Alas2 were among the most upregulated genes. We observed dysregulation of processes involved in development, mitochondrial function, cholesterol biosynthesis, and inflammation, all of which contribute to neurodevelopmental deterioration following GMH. This study is the first temporal transcriptome profile providing a comprehensive overview of the molecular mechanisms underlying brain injury following GMH, and it provides useful guidance in the search for therapeutic interventions.

Infection and inflammation are known risk factors for neonatal brain injury. Mycoplasma and Gram-positive bacteria, for which Toll-like receptor 2 (TLR2) plays a key role in recognition and inflammatory response, are among the most common pathogens in the perinatal period. Here, we report that systemic activation of TLR2 by Pam3CSK4 (P3C) increases neural tissue loss and demyelination induced by subsequent hypoxia–ischemia (HI) in neonatal mice. High-resolution respirometry of brain isolated mitochondria revealed that P3C suppresses ADP-induced oxidative phosphorylation, the main pathway of cellular energy production. The results suggest that infection and inflammation might contribute to HI-induced energy failure.

Cerebral palsy will affect nearly 10% of the 60 000 very low-birth-weight infants born in the United States in the next year, and an even greater percentage will display some form of permanent neurological impairment resulting from injury to the preterm brain. The 2008 Neurobiology of Disease in Children Symposium, held in conjunction with the 37th annual meeting of the Child Neurology Society, aimed to define current knowledge and to develop specific aims for future clinical, translational, and fundamental science. A complex interplay of both destructive and developmental forces is responsible for injury to the preterm brain. Advances in imaging and histology have implicated a variety of cell types, though preoligodendrocyte injury remains the focus. Research into different mechanisms of injury is facilitating new neuroprotective and rehabilitative interventions. A cooperative effort is necessary to translate basic research findings into clinically effective therapies and better care for these children.

We hypothesized that hypoxic preconditioning (PC) modifies the microvasculature in the immature brain and thereby affects the cerebral blood flow (CBF) during a subsequent hypoxic—ischemic (HI) insult. On postnatal day 6 rats were exposed to hypoxia (36°C, 8.0% O2) or normoxia for 3 h. Unilateral HI (unilateral carotid ligation and 8% hypoxia) was induced 24 h later. Cortical CBF was measured with the 14C-iodoantipyrine technique (at the end of HI) or with laser Doppler flowmetry (Perimed PF5001) before and during HI. At 0, 2, 8, and 24 h cerebral cortex was sampled and analyzed with gene arrays (Affymetrix 230 2.0). l-nitroarginine or vehicle was administrated before hypoxic PC or 30 mins before HI followed by CBF measurement (laser Doppler) during subsequent HI. Twenty-four hours after PC animals were perfusion-fixed and brains immunolabeled for von Willebrand factor and vascular density was determined by stereological quantification. The decrease in CBF during HI was attenuated significantly in PC versus control animals ( P < 0.01), as detected by both techniques. Several vascular genes (Angpt2, Adm, Apln, Vegf, Flt1, Kdr, Pdgfra, Agtrap, Adora2a, Ednra, serpine1, caveolin, Id1, Prrx1, Ero1l, Acvrl1, Egfl7, Nudt6, Angptl4, Anxa2, and NOS3) were upregulated and a few (Csrp2, Adora2b) were downregulated after PC. A significant increase in vascular density ( P < 0.05) was seen after PC. Nitric oxide synthase inhibition did not affect CBF during HI after PC. In conclusion, hypoxic PC upregulates vascular genes, increases vascular density and attenuates the decrease of CBF during a subsequent HI, which could contribute to tolerance.

Ischemia induces a complex response of differentially expressed genes in the brain. In order to understand the specific mechanisms of injury in the developing brain, it is important to obtain information on global changes in the transcriptome after neonatal hypoxia-ischemia. In this study, oligonucleotide arrays were used to investigate genomic changes at 2, 8, 24, and 72 hours after neonatal hypoxia-ischemia, which was induced in 9-day-old mice by left carotid artery ligation followed by hypoxia (10% O2). In total, 343 genes were differentially expressed in cortex, hippocampus, thalamus, and striatum 2 to 72 hours after hypoxia-ischemia, when comparing ipsilateral with contralateral hemispheres and with controls, using the significance analysis for microarrays. A total of 283 genes were upregulated and 60 were downregulated, and 94% of the genes had not previously been shown after neonatal hypoxia-ischemia. Genes related to transcription factors and metabolism had mostly upregulated transcripts, whereas most downregulated genes belonged to the categories of ion and vesicular transport and signal transduction. Genes involved in transcription, stress, and apoptosis were induced early after the insult, and many new genes that may play important roles in the pathophysiology of neonatal hypoxiaischemia were identified.

In a situation with normal CBF and without increased energy utilization, increased glucose utilization (CMRglc) can be a sign of impaired mitochondrial metabolism, which may be an early step in the injury cascade during reperfusion after hypoxia–ischemia (HI). Seven-day-old rats underwent unilateral carotid artery ligation and 70 minutes of HI. At 3, 6, 12, 24, and 48 or 72 hours after the insult, the CMRglc was measured by the 2-deoxyglucose method, and CBF by the iodoantipyrine method. These were compared with hematoxylin-eosin staining and microtubule-associated protein 2 (MAP 2) immunostaining in adjacent sections. In the ipsilateral hemisphere, there appeared regions with increased CMRglc compared with the contralateral hemisphere 3 to 12 hours after HI that also showed partial loss of MAP 2 immunostaining and early ischemic changes. These areas receded, leaving central glucose hypoutilizing areas with complete loss of MAP 2 immunostaining and histologic infarction, surrounded by only a rim of tissue with increased CMRglc. At 24 and 72 hours after the insult, no regions with increased CMRglc remained. Despite loss of MAP 2 immunostaining and histologic signs of infarction at 24 hours, cortical CBF was not reduced until 48 hours after HI, whereas the CBF in the caudate-putamen already was decreased compared with the contralateral side at 3 hours after HI. In conclusion, early reperfusion is characterized by glucose hyperutilizing areas in the cerebral cortex, followed by a secondary phase with low CMRglc and infarction.

Post-treatment with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 reduces hypoxic–ischemic brain injury in immature animals. To elucidate possible mechanisms, cerebral glucose utilization (CMRglc) and cerebral blood flow (CBF) were measured 1–5 h after hypoxia–ischemia and administration of MK-801 in 7-day-old rats. After 100 min of unilateral hypoxia–ischemia, half of the pups were injected with MK-801. CMRglc was assessed by the [14C]deoxyglucose (2-DG) method. The brains were analyzed either by autoradiography or for energy metabolites and chromatographic separation of 2-DG-6-phosphate and 2-DG. CBF was measured by the autoradiographic [14C]iodoantipyrine method. Mean CMRglc in the cerebral cortex was increased ipsilaterally after hypoxia–ischemia to 15 ± 3.3 μmol 100 g−1 min−1 ( p < 0.01) and areas with CMRglc >20 μmol 100 g−1 min−1 amounted to 8.0 ± 7.7 mm2 in the ipsilateral hemisphere compared with 1.2 ± 1.6 mm2 contralateral ( p < 0.001). Treatment with MK-801 decreased CMRglc bilaterally ( p < 0.05) and reduced ipsilateral areas with increased CMRglc by 64% ( p < 0.01). CBF was unaltered after hypoxia–ischemia and by MK-801 treatment. In conclusion, regional glucose hyper-utilization in the parietal cortex after hypoxia–ischemia was attenuated by MK-801; this may have relevance to the neuroprotective effect of NMDA-receptor antagonists in this model.

After 6–12 h of recovery from transient cerebral ischemia, the pyramidal cells of the hipppocampal CA1 region take up excessive amounts of calcium upon electrical stimulation, which has been suggested to be important for the development of delayed neuronal death. The aim of this study was to further characterize this enhanced calcium uptake with respect to time-course of development, relationship to neuronal damage, and amplitude of evoked field potentials as well as the dependency on N-methyl-d-aspartate (NMDA) and non-NMDA receptors. Adult Wistar rats were used and calcium-sensitive microelectrodes were placed in the stratum radiatum of the CA1 hippocampus for recording of the extracellular calcium concentration ([Ca2+]ec) during 20 min of ischemia and for 6 h of reflow. High-frequency stimulation of the perforant pathway elicited burst firing in CA1 and a transient decrease in [Ca2+]ec which reflects neuronal uptake. Shifts in [Ca2+]ec could not be evoked 0–1 h after ischemia. However, from 1–2 h burst firing could be evoked and the accompanying shift in [Ca2+]ec increased thereafter in amplitude with prolonged reflow, exceeded preischemic levels after 4 h, and reached 250 ± 116% (mean ± SD) of control after 6 h of reflow ( p < 0.05). The extracellular reference potential shift during electrical stimulation and the amplitude of evoked field potentials were still subnormal after 6 h [85 ± 25% and 83 ± 25%, respectively (mean ± SD)]. There was a significant correlation between the degree of stimulated calcium uptake at 6 h postischemia and the extent of CA1 damage evaluated 7 days after the ischemic insult ( r = 0.849; p < 0.001). The shifts in [Ca2+]ec were reduced by the NMDA antagonist MK-801 (0.5–2 mg/kg, i.v.) to approximately 50% of the initial level during both control and postischemic conditions ( p < 0.01). The non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[ F]quinoxaline (NBQX) (42 ± 13 mg/kg, i.p.; mean ± SD) decreased the amplitude of the evoked field potentials (to 30 ± 28% of control, p < 0.05) and completely abolished the evoked shifts in [Ca2+]ec. In conclusion, the uptake of calcium into CA1 pyramidal cells during electrical stimulation was enhanced already 4 h after ischemia in spite of the fact that other measures of excitability were subnormal. This calcium uptake correlated to the extent of CA1 pyramidal cell damage and was dependent on both NMDA and non-NMDA receptor activation.

Cerebral blood flow (CBF, by laser Doppler flowmetry) and extracellular cortical concentrations (by microdialysis) of adenosine, inosine, xanthine, hypoxanthine, and lactate were measured together with somatosensory evoked potentials (SEP) in chloralose-anaesthetized spontaneously hypertensive rats (SHR) during relative cerebral ischemia induced by hypotensive hemorrhage. Reduction of mean arterial blood pressure (MABP) to 40–50 mm Hg, which decreased SEP to about 50% of prebleeding control level, decreased CBF only to about 75% of control due to cerebrovascular “autoregulation.” A secondary, marked rise in cerebrovascular resistance (CVR) occurred after about 15 min in parallel with a striking increase in heart rate (after initial bradycardia). This late rise in heart rate is probably elicited by relative ischemia in medullary centers. The increase in CVR might indicate increased sympathetic nerve activity to the circle of Willis and large cerebral arteries. Cortical lactate increased initially but started to decline after about 30 min, and after 2 h it was not significantly higher than control. Cortical adenosine, inosine, hypoxanthine, and xanthine increased slowly and were significantly elevated after 50 min of hemorrhage. After 80 min, adenosine and inosine had returned to initial levels, while hypoxanthine and xanthine were further elevated. Despite the apparent partial recovery of metabolic disturbances during late hemorrhage, and with a blood flow maintained at 75% of resting control, SEP did not improve. It is suggested that the depression of SEP is not primarily caused by circulatory-metabolic derangements, but instead by activation of specific inhibitory systems.

The removal of glutamatergic afferents to CA1 by destruction of the CA3 region is known to protect CA1 pyramidal cells against 10 min of transient global ischemia. To investigate further the pathogenetic significance of glutamate, we measured the release of glutamate in intact and CA3-lesioned CA1 hippocampal tissue. In intact CA1 hippocampal tissue, glutamate increased sixfold during ischemia; in the CA3-lesioned CA1 region, however, glutamate only increased 1.4-fold during ischemia. To assess the neurotoxic potential of the ischemia-induced release of glutamate, we injected the same concentration of glutamate into the CA1 region as is released during ischemia in normal, CA3-lesioned, and ischemic CA1 tissue. We found that this particular concentration of glutamate was sufficient to destroy CA1 pyramids in the vicinity of the injection site in intact and CA3-lesioned CA1 tissue when administered during control (non-ischemic) conditions. In contrast, the same amount injected during ischemia in the CA3-lesioned CA1 region destroyed pyramidal cells in a widely distributed zone around the injection site in the CA1 region. It is concluded that the ischemia-induced damage of pyramidal cells in CA1 is dependent on glutamate release and intact innervation from CA3.

Brain ischemia was induced for 10 or 30 min by clamping the common carotid arteries in rabbits whose vertebral arteries had previously been electrocauterized. EEG and tissue content of high energy phosphates were used to verify the ischemic state and to evaluate the degree of postischemic recovery. Extracellular levels and total contents of amino acids were followed in the hippocampus during ischemia and 4 h of recirculation. At the end of a 30-min ischemic period, GABA had increased 250 times, glutamate 160 times, and aspartate and taurine 30 times in the extracellular phase. The levels returned to normal within 30 min of reflow. A delayed increase of extracellular phosphoethanolamine and ethanolamine peaked after 1–2 h of reflow. Ten minutes of ischemia elicited considerably smaller but similar effects. With respect to total amino acids in the hippocampus, glutamate and aspartate decreased to 30–50% of control while GABA appeared unaffected after 4 h of reflow. Alanine, valine, phenylalanine, leucine, and isoleucine increased severalfold. The importance of toxic extracellular levels of excitatory amino acids, as well as of high extracellular levels of inhibitory amino acids, are considered in relation to the pathophysiology of neuronal cell loss during cerebral ischemia.