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Neonatal hypoxic-ischemic brain injury (HIBI), resulting from impaired cerebral blood flow and oxygen delivery to the brain, affects at least 1.5 per 1,000 live births each year in the United States. Altered levels of cholesterol and cholesterol metabolites have been identified in brain tissue and in serum after adult brain injury such as traumatic brain injury and stroke. We hypothesized that there would be temporal and brain region-specific alterations in cholesterol and sterol precursors after neonatal HIBI.
Postnatal day 9 CD1 mouse pups were anesthetized with isoflurane and randomized to HIBI induced by carotid artery ligation or controls receiving sham surgery consisting of dissection without ligation (n=24/group). Pups were allowed to recover after surgery and then placed in a hypoxia chamber at 8% oxygen for HIBI or 21% for controls for 30 minutes. Each group was further divided into three sub-groups (n=8/group) for blood and brain tissue collection at 30 minutes, 24 hours, or 72 hours after injury. Brain tissue was dissected into four regions: cortex, cerebellum, striatum/thalamus, and hippocampus. For each region, protein was quantified by BCA assay, interleukin-6 (IL-6) levels were measured by ELISA as a marker of injury severity, and liquid chromatography mass spectrometry was performed to evaluate for the following sterols: cholesterol, desmosterol, 7-dehydrocholesterol, 8-dehydrocholesterol, and lanosterol. Levels were compared between HIBI and control groups at each time point and neuroanatomical region. Additionally, the four samples in each group demonstrating the most severe injury, as defined by IL-6 levels, were stratified as “severe injury”. Differences were analyzed with a two-sided Mann-Whitney test.
When assessing the entire cohort, no statistically significant differences were seen between HIBI and controls with respect to temporal or regional differences for any of the sterols measured. In animals with severe HIBI, however, cholesterol, 7-dehydrocholesterol, 8-dehydrocholesterol, and desmosterol were higher in the cortex at 24 hours after injury compared to controls. Additionally, desmosterol was also elevated in the cerebellum but decreased in the striatum 24 hours after injury in the severely injured animals.
Severe neonatal encephalopathy appears to be associated with alterations in cortical brain sterol levels, peaking around 24 hours after injury. Further defining the aberrations in brain metabolism in infants with HIBI could provide opportunities for not only diagnostic biomarkers but also the development of targeted therapies.
Dave, Amanda; Allen, Luke; Korade, Zeljka; and Peeples, Eric, "Evaluation of brain cholesterol metabolism after neonatal hypoxic-ischemic brain injury" (2021). Child Health Research Institute Pediatric Research Forum. 8.