Dysmaturation of Somatostatin Interneurons Following Umbilical Cord Occlusion in Preterm Fetal Sheep

Abstract

Introduction: Cerebral white matter injury is the most common neuropathology observed in preterm infants. However, there is increasing evidence that gray matter development also contributes to neurodevelopmental abnormalities. Fetal cerebral ischemia can lead to both neuronal and non-neuronal structural-functional abnormalities, but less is known about the specific effects on interneurons.

Objective: In this study we used a well-established animal model of fetal asphyxia in preterm fetal sheep to study neuropathological outcome. We used comprehensive stereological methods to investigate the total number of oligodendrocytes, neurons and somatostatin (STT) positive interneurons as well as 3D morphological analysis of STT cells 14 days following umbilical cord occlusion (UCO) in fetal sheep.

Materials and methods: Induction of asphyxia was performed by 25 min of complete UCO in five preterm fetal sheep (98-100 days gestational age). Seven, non-occluded twins served as controls. Quantification of the number of neurons (NeuN), STT interneurons and oligodendrocytes (Olig2, CNPase) was performed on fetal brain regions by applying optical fractionator method. A 3D morphological analysis of STT interneurons was performed using IMARIS software.

Results: The number of Olig2, NeuN, and STT positive cells were reduced in IGWM, caudate and putamen in UCO animals compared to controls. There were also fewer STT interneurons in the ventral part of the hippocampus, the subiculum and the entorhinal cortex in UCO group, while other parts of cortex were virtually unaffected (p > 0.05). Morphologically, STT positive interneurons showed a markedly immature structure, with shorter dendritic length and fewer dendritic branches in cortex, caudate, putamen, and subiculum in the UCO group compared with control group (p < 0.05).

Conclusion: The significant reduction in the total number of neurons and oligodendrocytes in several brain regions confirm previous studies showing susceptibility of both neuronal and non-neuronal cells following fetal asphyxia. However, in the cerebral cortex significant dysmaturation of STT positive neurons occurred in the absence of cell loss. This suggests an abnormal maturation pattern of GABAergic interneurons in the cerebral cortex, which might contribute to neurodevelopmental impairment in preterm infants and could implicate a novel target for neuroprotective therapies.

Keywords: GABA; interneurons; preterm; somatostatin; stereology.

FIGURE 1

One hemisphere of sheep brain that is cut into four blocks (top); Brain regions of interest in block B and block C. Scale bar = 6.5 mm.

FIGURE 2

The effect of umbilical cord occlusion (UCO) on the total number of somatostatin (STT) interneurons in block B regions of interest: (A) caudate; (B) putamen; (C) intragyral white matter (IGWM) (^∗p < 0.05, ^∗∗∗p < 0.001). (D) Illustration of counting the number of STT interneuron inside the unbiased counting frame on the light microscope connected to newCAST software with a 63X oil immersion lens. Mean ± SD. Scale bar = 15 μm.

FIGURE 3

The effect of UCO on the total number of STT interneurons in block C regions of interest: (A) entorhinal cortex, (B) subiculum, and (C) ventral DG. Mean ± SD. ^∗p < 0.05. DG, dentate gyrus.

FIGURE 4

Morphological analysis of STT interneurons showed a significant effect of UCO on the length and number of the STT dendrites in cortex, caudate, and putamen. Mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01.

FIGURE 5

Morphological analysis of STT interneurons showed a significant effect of UCO on the length of the STT dendrites in subiculum and EC. Mean ± SD. ^∗∗∗p < 0.001.

FIGURE 6

Branching pattern alterations of STT interneurons in cortex, caudate, putamen, subiculum, and EC using Sholl analysis in UCO and control groups. The number of branching intersections at various distances away from the cell soma was significantly lower in the UCO group vs. control group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Example of morphological analysis of STT interneuron in the putamen area of brain from control (left) and UCO (right) animals. Scale bar = 20 μm.

FIGURE 7

The number of Olig-2 cells was significantly lower in (A) IGWM, (B) caudate, and (C) putamen areas in UCO group compared to the control group. ^∗p < 0.05. (D) Olig-2 positive cells with dark brown soma. Scale bar = 10 μm.

FIGURE 8

Log transformed number of CNPase positive cells in the IGWM area of the UCO and the control groups. IGWM, intragyral white matter. ^∗p < 0.05 (left). Illustration of quantification of the number of CNPase cells in IGWM area of preterm fetal sheep (right). Scale bar = 10μm.

FIGURE 9

Effect of UCO on the number of NeuN positive neurons in IGWM, cortex, PVWM, putamen, and caudate. ^∗p < 0.05, ^∗∗p < 0.01.