298 - Modeling Neonatal Hypoxic-Ischemic Encephalopathy in Rats: A New Physiologically Relevant Approach

Publication Number: 4293.298

Salla-Maaria E. Alaollitervo, University of Turku, Helsinki, Uusimaa, Finland; Saeka Shimochi, University of Turku, Turku, Varsinais-Suomi, Finland; Emma Saha, Turku University Hospital, Naantali, Varsinais-Suomi, Finland; Varpu M. Laine, University of Turku, Mynämäki, Varsinais-Suomi, Finland; Vilhelmiina Parikka, University of Turku, Turku, Varsinais-Suomi, Finland

Background: Hypoxic-ischemic encephalopathy (HIE) resulting from perinatal asphyxia remains a leading cause of neonatal morbidity and mortality. Existing rodent models primarily induce unilateral cerebral injury, not fully replicating the systemic hypoxic-ischemic conditions observed in neonates.

Objective: To establish a physiologically relevant asphyxia-based model of global HIE in postnatal day (PND) 9-10 rats using controlled mechanical ventilation.

Design/Methods: PND 9-10 Sprague-Dawley rats, developmentally equivalent to term human infants, were anesthetized with subcutaneous (s.c.) medetomidine and ketamine, intubated with a 22G plastic IV cannula using a guiding wire and a small otoscope ear camera, and subsequently paralyzed with s.c. rocuronium. After a 10-minute stabilization period, pups underwent 30 minutes of global asphyxia consisting of controlled intermittent ventilation cycles: 1 minute of apnea followed by 1-2 minutes of low-rate ventilation. Blood gases and metabolic parameters were analyzed immediately post-insult, and brains were harvested 7 days later for histology and immunostaining.

For comparison, blood gases and metabolic profiles were obtained from PND 9 pups subjected to unilateral brain injury induced by permanent ligation of the left common carotid artery under isoflurane anesthesia followed by 2-hour hypoxia (8% O₂).

Results: The novel asphyxia model induced consistent systemic effects, including significant acidosis (pH 6.85 ± 0.12) with low HCO3 (13,9 ± 1,6 mmol/L) and base excess (BE -19,5 ± 2,9 mmol/L) together with elevated pCO2 (10,7 ± 2,8 kPa; ~80 mmHg) and lactate (13,2 ± 0,9 mmol/L). In the unilateral model, lactate (14,5 ± 1,3 mmol/L), HCO3 (13,8 ± 1,9 mmol/L) and BE (-11,3 ± 2,7 mmol/L) levels were indicative for systemic hypoxia. However, systemic acidosis was not observed (pH 7,36 ± 0,05) and pCO2 levels were low (3,2 ± 0,3 kPa; ~24 mmHg), likely reflecting the pups' ability to develop spontaneous hypocapnia through hyperventilation. Notably, pups in the new HIE model exhibited significant bradycardia during asphyxia. Preliminary immunostaining revealed morphological damage, with Fluoro-Jade C and Iba1 staining detecting selective bilateral neurodegeneration in hippocampal neurons.

Conclusion(s): The novel asphyxia-based model for neonatal HIE is feasible, induces systemic tissue hypoxia, and produces global hypoxic-ischemic injury, thereby accurately mimicking key features of term neonatal HIE. This model may provide a valuable experimental platform for elucidating HIE pathophysiology and for the preclinical testing of neuroprotective interventions.

Histological findings in the novel asphyxia model.
(A) H&E showing bilateral neuronal necrosis. DAPI (blue) and Fluoro-Jade C (green) merged image showing neuronal degeneration in the affected region. (B) Iba1 and GFAP immunostaining demonstrating microglial activation and reactive gliosis.