Background: Cellular targets of neonatal hypoxia-ischemia (HI) include both oligodendrocyte and neuronal lineages with differences in the patterns of vulnerable cells depending upon the developmental stage at which the injury occurs. Injury to the developing white matter is a characteristic feature of human preterm brain injury. Data are accumulating, however, for neuronal injury in the developing cerebral cortex. In the most widely used rodent model of preterm HI brain injury, conflicting data have been reported regarding the sensitivity of subplate neurons to early neonatal HI, with some reports of selective vulnerability and others that find no increased loss of subplate neurons in comparison with other cortical layers. Methods used to identify subplate neurons and quantify their numbers vary across studies. Objective: To use recently developed cortical layer-specific markers quantified with definitive stereologic methods to determine the magnitude and specificity of subplate neuron cell loss following neonatal HI in a rodent model. Methods: Postnatal day 2 (P2) rats underwent right common carotid artery coagulation followed by 2-3 h of hypoxia (5.6% oxygen). Categorically moderately injured brains were stained with subplate and cortical layer III-V markers (Complexin3 and Foxp1, respectively) at P8 and P21 (Foxp1 only). An Optical Fractionator was used to quantify subplate and middle/lower cortical neuronal numbers and these were compared across groups (naive control, hypoxia hemisphere, and HI hemisphere). Results: Following HI at P2 in rats, the total Complexin3-expressing subplate neuron number decreases significantly in the HI hemisphere compared with naive controls or hypoxia alone (HI vs. control 26,747 ± 7,952 vs. 35,468 ± 8,029, p = 0.04; HI vs. hypoxia, 26,747 ± 7,952 vs. 40,439 ± 7,363, p = 0.003). In contrast, the total Foxp1-expressing layer III-V cell number did not differ across the 3 conditions at P8 (HI vs. control 1,195,085 ± 436,609 vs. 1,234,640 ± 178,540, p = 0.19; HI vs. hypoxia, 1,195,085 ± 436,609 vs. 1,289,195 ± 468,941, p = 0.35) and at P21 (HI vs. control 1,265,190 ± 48,089 vs. 1,195,632 ± 26,912, p = 0.19; HI vs. hypoxia, 1,265,190 ± 48,089 vs. 1,309,563 ± 41,669, p = 0.49). Conclusions: There is significant biological variability inherent in both the subplate neuron cell number and the pattern and severity of cortical injury following HI at P2 in rats. Despite this variability, the subplate neuron cell number is lower following P2 HI in animals with mild or moderate cortical injury, whereas the middle-to-lower-layer cortical neuronal number is unchanged. In more severe cases, neurons are lost from the lower cortical layers, suggesting a relative vulnerability of subplate neurons.
Inhibition of Calcium/Calmodulin-Dependent Protein Kinase Kinase β Is Detrimental in Hypoxia–Ischemia Neonatal Brain Injury