Unbiased Quantification of Subplate Neuron Loss following Neonatal Hypoxia-Ischemia in a Rat Model

Abstract

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.

Keywords: Brain injury; Cortical layer-specific marker; Optical Fractionator; Premature newborn.

Figure 1. Subplate markers Nurr1 and CTGF colabel some cells with Cplx3

P8 rat control brain stained with subplate markers Complexin3 (green) and Nurr1 (red) (panel A) or Complexin3 (green) and CTGF (red) (panel B) show cells positive for both or either markers. Scale bar: 100um. Corresponding panels (A′ and B′) show a close up of single positive and double positive cells. Scale bar: 10 um.

Figure 2. Histological assessment of moderate cortical injury following hypoxia ischemia in P8 rat

Cresyl violet stained coronal sections of the three examined conditions: naive control (A), hypoxia (contralateral to injury, B), hypoxia-ischemia (ipsilateral to injury, C). Scale bar: 1mm. Corresponding panels (A′, B′, C′) show a close up of the areas indicated by black boxes. Comparable regions stained for Complexin3 with DAB show normal subplate (D), thinning subplate in hypoxia (E), and patches of subplate cell loss in HI (F). Scale bar: 200um for A′–F.

Figure 3. Regions of interest and counting frames for stereological quantification in P8 rat

DAB staining with subplate marker, Complexin3, (A) and layer III–V maker, Foxp1, (B). Contours in red indicate regions of interest used for analysis. Scale bar: 1mm. Example of a Complexin3+ cell (C, counting frame: 40umx40um) and Foxp1+ cell (D, counting frame: 20umx20um) counted using the Optical Fractionator probe.

Figure 4. Quantification of Complexin3 and Foxp1 across injury groups at P8

Complexin3+ subplate population in HI group is significantly decreased compared to control and hypoxia group (A, p =0.0177 and p= 0.0015). Foxp1+ cell numbers are not significantly different between injury groups (B). Hemispheric (right/left) ratios of Complexin3 and Foxp1 counts show a greater variability in the injured groups, compared to controls (C). Complexin3 cell counts in HI hemisphere are negatively correlated with infarct volume as defined by (infarct volume = (hypoxia - hypoxia-ischemia)/hypoxia) (D, r²=0.82, p=0.0017).

Figure 5. Quantification of Foxp1 across injury groups at P21

Foxp1+ cell numbers are not significantly different between injury groups (A). Hemispheric ratios (right/left) of Foxp1 counts have low variability between control and hypoxia (B). Foxp1 cell counts in HI hemisphere are not significantly correlated with infarct volume as defined by (infarct volume = (hypoxia - hypoxia-ischemia)/hypoxia) (C, r²=0.56, p=0.1418).