PMC6458245_01
Histological section of lung showing the presence of abundant endoalveolar and endobronchial corneal scales into the alveoli. Staining: hematoxylin/eosin; Scale bar: 50 mum.
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F1
The lung sections highlighted the presence of abundant endoalveolar and endobronchial corneal scales induced by the aspiration of amniotic fluid, with meconium residues; several areas of emphysema, probably of a compensatory nature, were also detected especially in the right lung (Figure 1).
(Right) Schematic representation of the brainstem sampling: I = ponto-mesencephalic specimen, including the upper third of the pons and the adjacent portion of mesencephalon; II = caudal pontine specimen; III = medulla oblongata specimen, including the obex; IV = sample extending from the caudal pars of the medulla oblongata to the rostral spinal cord. (Left) schematic representative histological sections obtained from the four specimens, indicating the main nuclei and structures to be examined.
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F2
The protocol drawn up by the "Lino Rossi" Research Center of the Milan University in the context of the aforementioned Italian law, states that four specimens are to be taken from the brainstem after the routine fixation (as shown in Figure 2, at the right). Figure 2, on the left, shows representative histological sections obtained from the above-described specimens, indicating the main nuclei and structures to be examined, given their frequent involvement in sudden perinatal deaths in terms of delayed development (hypoplasia/agenesis). All the other main structures (represented in Figure 2, on the left) showed a normal cytoarchitecture, including the Kolliker-Fuse nucleus (KFN) and the pre-Botzinger complex (preBotC), both determinant neuron groups for respiratory pattern generation.
An image series related to the retrotrapezoid/parafacial respiratory group (RTN/pFRG). At the top, on the right: ventral brainstem schematic image with the indication (red arrow) of the optimal sampling for the examination of the human RTN/pFRG in the caudal pons; on the left: whole histological section at this level with the indication of the RTN/pFRG localization. (A) Histological section of medulla oblongata showing in the circled area the normal cytoarchitecture of the RTN/pFRG, adjacent to the facial nerve (fn) in an age-matched control case. (B) Severe hypoplasia of the RTN/pFRG observed in this case. (C) Cluster of PHOX2B immunoreactive neurons identifier of the RTN in the control case. (D) Lack of PHOX2B immunoreactivity in the RTN region. (A,B) Staining: Kluver-Barrera; (C,D) Staining: PHOX2B immunohistochemistry. Scale bar (A-D): 100 mum.
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F3
The histological analysis of the brainstem showed severe hypoplasia of the retrotrapezoid/parafacial respiratory group (RTN/pFRG) in the caudal pons when compared with the normal structure of this complex in the control case (Figures 3A,B). Intensely PHOX2B immunoreactive nuclei were present in fact only in the neurons of the control case, allowing to easily distinguish the RTN from the overlying parafacial neurons (Figure 3C), while no PHOX2B immunoreactivity was highlighted in the RTN region of the case object of this exposure (Figure 3D).
An image series related to the arcuate nucleus (AN). At the top, on the right: ventral brainstem schematic image with the indication (red arrow) of the optimal sampling for the examination of the human AN in the medulla oblongata (at the obex level); on the left: whole histological section at this level with the indication of the AN localization. (A) Histological section showing (red arrows) the normal structure of the AN at the ventral medullary surface in an age-matched control case. (B) Severe hypoplasia of the AN observed in this case. (A,B) staining: Kluver-Barrera; Scale bar (A,B): 100 mum.
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F4
In addition, a severe hypoplasia of the arcuate nucleus in the medulla oblongata was observed (Figure 4).