Chronic fetal hypoxia affects axonal maturation in guinea pigs during development: A longitudinal diffusion tensor imaging and T2 mapping study

Abstract

Purpose: To investigate the impact of chronic hypoxia on neonatal brains, and follow developmental alterations and adaptations noninvasively in a guinea pig model. Chronic hypoxemia is the prime cause of fetal brain injury and long-term sequelae such as neurodevelopmental compromise, seizures, and cerebral palsy.

Materials and methods: Thirty guinea pigs underwent either normoxic and hypoxemic conditions during the critical stage of brain development (0.7 gestation) and studied prenatally (n = 16) or perinatally (n = 14). Fourteen newborns (7 hypoxia and 7 normoxia group) were scanned longitudinally to characterize physiological and morphological alterations, and axonal myelination and injury using in vivo diffusion tensor imaging (DTI), T2 mapping, and T2 -weighted magnetic resonance imaging (MRI). Sixteen fetuses (8 hypoxia and 8 normoxia) were studied ex vivo to assess hypoxia-induced neuronal injury/loss using Nissl staining and quantitative reverse transcriptase polymerase chain reaction methods.

Results: Developmental brains in the hypoxia group showed lower fractional anisotropy in the corpus callosum (-12%, P = 0.02) and lower T2 values in the hippocampus (-16%, P = 0.003) compared with the normoxia group with no differences in the cortex (P > 0.07), indicating vulnerability of the hippocampus and cerebral white matter during early development. Fetal guinea pig brains with chronic hypoxia demonstrated an over 10-fold increase in expression levels of hypoxia index genes such as erythropoietin and HIF-1α, and an over 40% reduction in neuronal density, confirming prenatal brain damage.

Conclusion: In vivo MRI measurement, such as DTI and T2 mapping, provides quantitative parameters to characterize neurodevelopmental abnormalities and to monitor the impact of prenatal insult on the postnatal brain maturation of guinea pigs.

Keywords: DTI; T2; brain development; fetal hypoxia; guinea pig; prenatal brain injury.

Figure 1

Effect of chronic fetal hypoxia on neuronal density and brain injury markers in the fetal brain. Photomicrographs of Nissle stained coronal sections of the fetal guinea pig brains in the NMX (a, left) and HPX (a, right) groups at the interaural level of 6.72–5.40 mm. Neuronal structures with RNA are shown in blue. (b) Neuronal density in cingulate, hippocampus, and cortex. Neuronal density was measured from histological sections of each region by counting the number of Nissl stained cells per square millimeter. (c) Expression levels of mRNA for brain injury indices (EPO and H1F-1α) in hippocampus. (*) indicates significant differences (p < 0.05) between the HPX and NMX groups.

Figure 2

MR images of guinea pig brains in the NMX and HPX groups at postnatal day 1 (P1) and 28 (P28). (a) High resolution T₂-weighted images during brain development of guinea pigs. The ROI of the hippocampus and a double-headed arrow indicating maximum brain width measurements are shown in the NMX P1 image (top, leftmost). The ROIs for T₂ sampling are shown in the ‘HPX P28’ image: 1) cingulate, 2) hippocampus and 3) cortex, and three double-headed blue arrows indicate areas where cortical thickness was measured. (b) RGB fractional anisotropy (FA) maps corresponding to high resolution T₂-weighted images in (a). An ROI of the corpus callosum is shown in the P28 HPX image (middle, rightmost). Colors indicate different diffusion directions: red for x (left to right), green for y (caudal-rostral) and blue for z (anterior-posterior). (c) Corresponding T₂ maps at P1 and P28 of a normoxic and a hypoxic brains. The gray scale bar on the right indicates T₂ values between 10–100 ms.

Figure 3

Comparisons of (a) FA and (b) MD values from the corpus callosum of guinea pig brains between the NMX (□) and HPX (■) groups at postnatal days 1 (P1), 7 (P7), 28 (P28) and 42 (P42). (*) indicates significant differences (p < 0.05) between the HPX and NMX groups at each age. (#) and (†) indicate significant longitudinal differences (p < 0.05) between P1 and P28 in the HPX and NMX groups, respectively.

Figure 4

Comparisons of T₂ values in (a) the hippocampus and (b) the cortex of guinea pigs brains between the NMX (□) and HPX (■) groups at postnatal days 1 (P1), 7 (P7), 28 (P28) and 42 (P42). (*) indicates significant differences (p < 0.05) between the HPX and NMX groups at each age.

Figure 5

Comparisons of structural changes of guinea pig brains in the NMX (□) and HPX (■) groups at postnatal days 1 (P1), 7 (P7), 28 (P28) and 42 (P42). Time course of (a) body weight, (b) brain width, (c) hippocampal size and (d) cortical thickness are shown from P1 to P42. (*) indicates significant differences (p < 0.05) between the HPX and NMX groups at each age. (#) and (†) indicate significant longitudinal differences (p < 0.05) between P1 and P7 to P28 in the HPX and NMX groups, respectively.