Mannitol as a crystalloid substitute for colloid oncotic support in the respiratory active working heart-brainstem preparation

The working heart-brainstem preparation (WHBP) is an arterially perfused in situ rodent model that requires an osmotically or oncotically active perfusate additive to limit edema and maintain stable central respiratory motor output. Ficoll-70 at 1.25% (w/v) is widely used for this purpose, but at this concentration it generates only modest colloid osmotic pressure (∼3-4 mmHg) and presents practical limitations, including batch variability, endotoxin risk, and high cost. We asked whether mannitol at 0.85% (w/v), a small-molecule crystalloid osmolyte and hydroxyl-radical scavenger with an established clinical safety profile, can substitute for Ficoll-70 while preserving WHBP viability. Adult mouse WHBPs (n = 7 Ficoll; n = 9 mannitol) were perfused for 1 h with Krebs-based perfusate containing either 1.25% Ficoll-70 or 0.85% mannitol. Preparations were assessed for phrenic and vagus nerve activity, perfusion pressure, whole-preparation weight gain, brain tissue weight, and single-neuron recording capability. Phrenic discharge frequency increased over time in both groups (Ficoll: 0.15 → 0.49 Hz; mannitol: 0.24 → 0.45 Hz), and amplitude of the integrated phrenic burst did not differ significantly between additives. Perfusion pressure rose modestly over 1 h (Ficoll: 67.7 → 76.7 mmHg; mannitol: 67.2 → 74.1 mmHg), with no significant between-group difference. Whole-preparation weight increased in both conditions, indicating tissue water uptake during perfusion. Brainstem-cerebellum wet weight after 1 h was similar with both perfusates (0.140 ± 0.023 g, Ficoll; 0.124 ± 0.020 g, mannitol; p = 0.18). Juxtacellular recordings from preBötzinger complex neurons confirmed that single-unit activity can be obtained reliably in mannitol-perfused preparations for more than 2 h. Moreover, post-inspiratory activity of the vagus nerve is preserved in mannitol-perfused preparations. Together, these data indicate that 0.85% mannitol maintains WHBP cardiorespiratory function and tissue water balance to a degree comparable with 1.25% Ficoll-70, while providing a lower-cost, endotoxin-free alternative. The use of mannitol as a crystalloid perfusate additive may therefore facilitate wider and more reproducible application of the WHBP and related arterially perfused in situ preparations in integrative physiology and pharmacology.