Characterisation of putative retrotrapezoid nucleus (RTN) chemoreceptor neurons in the adult human brainstem

Human tissue selection

Post-mortem, formalin-fixed brainstem specimens from two adult human cases (76-year-old female, and 87-year-old male; Supplementary Table 1), were sourced from the Sydney Brain Bank through regional brain donor programs in Sydney, Australia. Specimens were acquired from asymptomatic individuals exhibiting solely age-related brain changes and devoid of significant neuropathology. Exclusion criteria included documented respiratory drive disorders, post-mortem delays exceeding 72 h and formalin fixation exceeding ten years. The demographic and autopsy characteristics are presented in Supplementary Table 1.

Brainstem tissue blocks spanning from the rostral medulla oblongata (Obex + 12 mm) to the caudal pons (Obex + 18 mm) were selected (Fig. 1), as this region contains the 7N and is expected to include the hRTN. This Obex range was guided by a human brainstem atlas (Paxinos et al. 2020).

Fig. 1figure 1

Tissue sectioning and parafacial area localisation (a) Sagittal schematic of the adult human brainstem and thalamus indicating the angle of tissue sectioning and location spanning from Obex (indicated by blue arrow) + 12 to + 18 mm (between red lines). b and c Representative H&E stained caudal (Obex + 14 mm) and rostral (Obex + 16 mm) hemi-sections overlaid with brainstem schematics. b Caudally, the parafacial (red outline) is encapsulated by a region medial to sp5 and Sp5O, ventral to 7N, lateral to the SO and overlapping with C1 or A5 neurons (blue outline). c Rostrally, the parafacial area is medial to 7n, ventral to 7N, lateral to MSO and overlaps with C1 or A5 neurons (blue outline). Adapted from (Paxinos et al. 2020)

Tissue sectioning

Ten-micron serial sections were cut from 2–3 mm thick transverse oriented FFPE brainstem blocks, using a rotary microtome. We collected approximately 700 sections per case, extending rostrocaudally from Obex + 18 to + 12 mm (Fig. 1). One in every thirty serial Sects. (300 μm apart) was stained with Hematoxylin & Eosin (H&E), to obtain representative sections containing the 7N. After mapping the full extent of the 7N in H&E sections, four of every thirty serial sections were selected for immunohistochemistry using antibodies against ChAT, Phox2b + TH, Phox2b + PACAP and Phox2b + galanin, respectively (Supplementary Table 2).

Immunohistochemistry (IHC) protocol for human FFPE brain tissue

The IHC protocol was adapted from our previous methods (Huang et al. 2017), with necessary optimisations for Phox2b detailed in Supplementary Material 1. Antigen retrieval was performed using heat-induced epitope retrieval (HIER) with citrate buffer (pH 6, #S2369, Agilent DAKO) for 15 min. After a 2 h block in 10% normal donkey serum (NDS) in tris buffered saline (TBS) with 0.3% Tween 20, sections were incubated overnight with goat anti-ChAT followed by a 2 h incubation with biotinylated secondary antibody, in 2% NDS /TBS with 0.1% Tween 20 (Table 2). Signal amplification used the Vectastain Elite ABC HRP Kit (#PK-6100, Vector Laboratories), and visualisation was achieved using 3,3’-diaminobenzidine (DAB Substrate Kit, #SK-4100, Vector Laboratories).

Table 2 Primary and secondary antibodies used for immunohistochemistry (IHC).

For dual labelling (Phox2b + TH, Phox2b + PACAP, or Phox2b + galanin), Phox2b was detected first and visualised using DAB with nickel enhancement (black) (#SK-4100, Vector Laboratories). In the sequential round of immunohistochemistry, the tissue was incubated with a primary antibody against TH, PACAP, or galanin, followed by the corresponding secondary antibody and DAB (brown) visualisation. After dehydration and clearing, sections were coverslipped using dibutylphthalate polystyrene xylene (DPX). Antibody details and concentrations are listed in Table 2.

Microscopy and imaging

Slides were digitised using a slide scanner (Aperio AT2, Leica BioSystems) at 20 × magnification. The scanned files were viewed using QuPath Software (Version 0.2.3) (Bankhead et al. 2017). Coronal hemi-sections approximating from Obex + 12 to + 18 mm were assessed.

The parafacial area was defined as follows: rostrocaudally, spans from the rostral ventrolateral medulla to the caudal ventrolateral pons, located adjacent to the facial motor nucleus (Obex + 13 to + 17 mm),dorsoventrally and mediolaterally, it is ventral to the spinal trigeminal nucleus, oral part (Sp5O), dorsal to ventral medullary surface, lateral to the intermediate reticular nucleus (IRt) and inferior olive (IO), and medial to the facial nerve (7n) (Fig. 1). Schematic diagrams were drawn using Adobe Illustrator 2024 (Adobe Inc., San Jose, CA, USA).

Quantitative analyses

The cell diameter, and area of the cell soma and nucleus of RTN neurons, were measured using QuPath tools (See details in Supplementary Material 2). This data was collated using Microsoft Excel v14.0 (2010, Microsoft, USA), and exported into GraphPad Prism (Version 9, CA, USA).

Neurons with the following neurochemical signatures were counted:

Phox2b immunoreactivity (-ir) parafacial neurons, located ventrolateral to the 7N, as:

Phox2b-ir RTN neurons: Phox2b + nucleus, TH- and ChAT- cytoplasm,

PACAP + /Phox2b + parafacial neurons and PACAP-/Phox2b + parafacial neurons,

Galanin + /Phox2b- parafacial neurons and galanin-/Phox2b + parafacial neurons,

7N neurons as ChAT + cytoplasm

C1 neurons: Phox2b + nucleus, TH + cytoplasm (Kang et al. 2007; Agostinelli et al. 2023),

A5 neurons: Phox2b- nucleus, TH + cytoplasm (Kang et al. 2007; Agostinelli et al. 2023).

Phox2b-negative RTN neurons were not investigated in this study. The RTN neurons referred in this study were Phox2b-ir RTN neuron. Number of RTN neurons was calculated by applying the formula derived from (Abercrombie 1946):

$$ \begin Phox2b - irRTN}Cell}Count}\left( N \right)\, \hfill \\ = \,RTN(Phox2b - ir)\, hemi\sec tion \,cell \,count(n) \times \frac} \times 2 \times 30 \hfill \\ \end $$

N, Total Phox2b-ir RTN cell count; n, RTN cell count/hemi-section; T, section thickness; D, average diameter of the cell.

Morphological data from the 2 adult cases was averaged and presented as mean ± standard deviation. No statistical analysis was performed due to the study being an anatomical description of the hRTN, without comparison between a control and experimental group.

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