Neuropeptides in the developing human hippocampus under hypoxic-ischemic conditions

Abstract

The perinatal period, sensitive for newborn survival, is also one of the most critical moments in human brain development. Perinatal hypoxia due to reduced blood supply to the brain (ischemia) is one of the main causes of neonatal mortality. Brain damage caused by perinatal hypoxia-ischemia (HI) can lead to neuro- and psychological disorders. However, its impact seems to be region-dependent, with the hippocampus being one of the most affected areas. Among the neuronal populations of the hippocampus, some interneuron groups - such as somatostatin- or neuropeptide Y-expressing neurons - seem to be particularly vulnerable. The limited information available about the effects of HI in the hippocampus comes mainly from animal models and adult human studies. This article presents an immunohistochemical analysis of somatostatin (SOM) and neuropeptide Y (NPY) expression in the developing human hippocampus after perinatal HI. Two rostrocaudal sections of the body of the hippocampus were analysed, and the number of immunostained cells in the polymorphic layer of the dentate gyrus (DG) and the pyramidal cell layer and stratum oriens of the CA3, CA2 and CA1 fields of the hippocampus proper were quantified. The results showed a lower density of both neuropeptides in hypoxic compared to control cases. In the HI group, the number of SOM-immunoreactive cell bodies was statistically significantly lower in the pyramidal cell layer and stratum oriens of CA1, while the number of NPY-expressing neurons was statistically lower in the pyramidal cell layer of CA2. Besides, the number of SOM-expressing neurons was significantly higher in the stratum oriens of CA1 compared to that in CA2. In sum, we observed a different vulnerability of SOM- and NPY-containing neurons in the developing human hippocampus following perinatal HI damage. Our results could contribute to a better understanding of the behaviour of these neuronal populations under stressful conditions during the perinatal period.

Keywords: brain development; hippocampus; hypoxia-ischemia; neuropeptide Y; somatostatin.

FIGURE 1

Negative controls for the antibodies used in this study. (a): The primary antibodies (goat anti‐SOM and rabbit anti‐NPY) were omitted from their respective incubation baths, and sections were reacted with the respective biotinylated secondary antibodies and streptavidin before being counterstained with thionin. (b): Section incubated with primary antibody goat anti‐SOM followed by a biotinylated goat anti‐rabbit antibody and streptavidin. No immunoreaction was detected. See also controls carried out for the same antibodies in a previous work (Cebada‐Sánchez et al., 2014). Abbreviations: DG: dentate gyrus; CA1: ammonic hippocampal field 1; CA2: ammonic hippocampal field 2. Scale bar: 200 µm

FIGURE 2

Photographs of different fields of the hippocampus of case 6 (HYG). Clear cell loss can be observed (dashed line), mainly in the granular layer of the DG and in the pyramidal layer. (a and b): Nissl‐staining of DG (a) and ammonic fields (b). (c and d): SOM‐IR counterstained with thionin in the DG. The arrows indicate SOM‐IR cells in the polymorphic layer of the DG. (e): SOM‐IR counterstained with thionin in CA2 and CA1. The arrows indicate SOM‐IR cells in the pyramidal layer of CA1. (f): High‐power magnification of image (e). (g): NPY‐IR counterstained with thionin in CA2 and CA1. The arrows indicate NPY‐IR cells in the pyramidal layer of CA1. (h): High‐power magnification of image G. Abbreviations: DG: dentate gyrus; ml: molecular layer; gl: granular layer; pl: polymorphic layer; py: pyramidal layer; so: stratum oriens; CA1: ammonic hippocampal field 1; CA2: ammonic hippocampal field 2. Scale bar: 200 µm

FIGURE 3

Analysis of SOM‐IR and NPY‐IR cells in the polymorphic layer of the DG. Histograms show the mean value (±SEM) of SOM‐IR and NPY‐IR neuron density (cells/mm²) in CG (white bar) and HYG (dark bar)

FIGURE 4

SOM‐IR and NPY‐IR cell density (mean ± SEM) in the pyramidal layer of the ammonic fields. (a and b) SOM‐IR (a) and NPY‐IR (b) cell density by groups. (c–e) SOM‐IR and NPY‐IR cell density by area, (c) CA3, (d) CA2, and (e) CA1. White bar: CG; black bar: HYG. Significance *: p < 0.05. Abbreviations: CG: control group. HYG: hypoxia–ischemia group

FIGURE 5

SOM‐IR and NPY‐IR cell density (mean ± SEM) in the stratum oriens. (a) and (b) SOM‐IR (a) and NPY‐IR (b) cell density by groups. (c–e) SOM‐IR and NPY‐IR cell density by area, (c) CA3, (d) CA2, and (e) CA1. White bar: CG; black bar: HYG. Significance *: p < 0.05. Abbreviations: CG: control group. HYG: hypoxia–ischemia group

FIGURE 6

Distribution of SOM (a–d) and NPY (e–f) in the DG in both experimental groups. (a) SOM‐expressing neurons in the polymorphic layer of DG (case 4, CG). (b) High‐power magnification of the image squared in (a). (c) Distribution of SOM‐IR neurons in the HYG (case 6). (d) Magnification of the area squared in (c). (e): NPY‐IR neurons in the polymorphic layer of DG (case 2, CG). (f): Distribution of NPY‐expressing neurons in case 10 (HYG). Abbreviations: DG: dentate gyrus; ml: molecular layer; gl: granular layer; pl: polymorphic layer. Scale bar: 200 µm

FIGURE 7

SOM‐IR cells in the stratum oriens and pyramidal layer of CA2 and CA1. (a–c) Case 2, CG. (b) Magnification of area limited by square 1 in (a). (c) Magnification of area limited by square 2 in (b). (d–e): Case 10 (HYG). (e): Magnification of area limited by square 3 in (a). (f): Magnification of area limited by square 4 in (a). Abbreviations: CA1: ammonic hippocampal field 1; CA2: ammonic hippocampal field. Scale bar: 200 µm

FIGURE 8

NPY‐IR cells in the stratum oriens and pyramidal layer of CA2 and CA1. (a–c) Case 4 (CG). (b) Magnification of area limited by square 1 in (a). (c): Magnification of area limited by square 2 in (a). (d–e): Case 10 (HYG). (e) Magnification of area limited by square 3 in (a). (f) Magnification of area limited by square 4 in (e). Abbreviations: CA1: ammonic hippocampal field 1; CA2: ammonic hippocampal field. Scale bar: 200 µm