Biomarkers for Early Detection and Therapeutic Monitoring of Abnormal Brain Development in Mild Fetal Growth Restriction
Description
Fetal growth restriction (FGR), driven by intrauterine hypoperfusion, delays brain development and heightens the risk of neurodevelopmental disorders. Nonetheless, current diagnostic strategies rarely capture the subtle neuropathology that emerges in mild FGR. To overcome this limitation, we employed an innovative rodent model that replicates mild FGR through gradual and chronic intrauterine hypoperfusion, mirroring clinical conditions overlooked by conventional severe or acute FGR models. Global proteomics of cerebrospinal fluid identified Alpha-2-Macroglobulin, Neuroserpin, CD200, and Polyubiquitin-B as biomarkers correlated with birth weight and persisting postnatally. Their expression reflected changes in brain tissue and serum, was linked to behavioral deficits, and partially recovered under mesenchymal stem/stromal cell treatment—indicating potential for therapeutic monitoring. Notably, Neuroserpin, brain-specific, emerged as a robust indicator of FGR-related neurodevelopmental impairment. This study is the first to propose low-invasive serum biomarkers for early postnatal detection of mild FGR-induced brain abnormalities, enabling neonatal screening, targeted interventions, and improved long-term outcomes.
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All animal experiments were approved by the Nagoya University Animal Experiment Committee (Nagoya, Aichi Prefecture, Japan; Approval Numbers: 26128, 27191, 28002, 29015) and conducted in accordance with the Regulations on Animal Experiments at Nagoya University. All experiments were performed in a double-blind fashion and in accordance with the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines for the care and use of laboratory animals (88). All sample collection was performed under isoflurane anesthesia, and all efforts were made to minimize the number of animals used and their suffering. The number of animals was kept to the minimum required to achieve statistical significance. Pregnant Sprague-Dawley rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). All dams were housed individually in cages under controlled conditions (temperature: 22 ± 1°C, humidity: 50 ± 5%) with a 12-hour light/12-hour dark cycle and ad libitum access to food and water. In the study design A (Fig. 8), the dams were randomly assigned to Control (non-treated, n = 8), Sham (surgical procedure without hypoperfusion, n = 14), or Intrauterine hypoperfusion (ameroid constrictor-attached, n = 19) groups. Postnatally, thirty pups from Control, twenty-six from Sham, and twenty-seven from Intrauterine Hypoperfusion (FGR group) were used with low birth body weight below FGR threshold (FGR group). Control pups were used to calculate FGR model threshold, with their organs being used for another research project. From Sham and Intrauterine hypoperfusion groups, twenty pups each were used for proteomic analysis at PNDs 4 and 5 (n = 10/group/day), forty-five pups each for Western blotting at PNDs 4, 5, 7, 10, and 14 (n = 15/group), and ten pups each for histological analysis at PND 10 (n = 10/group). In the study design B (Fig. 8), dams were randomly assigned to Sham (n = 10) or Intrauterine hypoperfusion (n = 26) groups, with the latter split into vehicle-treated (n = 13) and MSC-treated (n = 13) subgroups. Postnatally, fouty-six pups were allocated to Sham and 109 to Intrauterine hypoperfusion (61 vehicle-treated, 48 MSC-treated). Sixty-five pups from Sham, vehicle-treated non-FGR (vehicle A), vehicle-treated FGR (vehicle B), and MSC-treated FGR groups underwent behavioral tests at PNDs 8-11, 30-31, and 140-141 (n = 7-11/group). Also, ninety pups from Sham, vehicle-treated FGR, and MSC-treated FGR groups were used for Western blotting at PNDs 5, 7, 10, and 14 (n = 10-20/group). The minimal sample size was calculated to achieve 80% power of testing with an α error rate of 5%, based on preliminary experiments that assumed an effect size of 1.5 in the behavioral test, the primary endpoint.
Institutions
- Sanyoonoda Shiritsu Yamaguchi Tokyo Rika DaigakuYamaguchi, Sanyoonoda