Introduction
Neuroinflammation is regulated by dynamic interactions between microglia and astrocytes and plays a crucial role in brain development. Increasing evidence highlights the microbiota–gut–brain axis as a key modulator of neuroinflammatory processes and neurodevelopment. Modulation of maternal gut microbiota during pregnancy may therefore represent a promising therapeutic strategy for improving offspring brain health.
Aims/objectives
This study investigated the impact of maternal gut dysbiosis on offspring neuroinflammation and neurodevelopment and evaluated the potential protective effects of probiotic supplementation.
Methods
Experiments were conducted on Wistar rat offspring (n=100) derived from dams assigned to four gestational treatment groups: control, antibiotics, probiotics, and antibiotics+probiotics. Animals were housed in the Biological Service Unit under standard conditions, with ad libitum access to food and water. All procedures were approved by the Ethics Committee of the Cantacuzino National Military Medical Institute for Research and Development (No. 319/2023) and by the veterinary health authority (No. 37/23.08.2023).
Maternal gut dysbiosis was induced by administering an antibiotic cocktail (ampicillin, vancomycin, neomycin, and meropenem) from embryonic day E11. A multi-strain probiotic was administered throughout pregnancy.
On postnatal day 6, pups were exposed to either perinatal asphyxia (PA) or normoxia (N), resulting in eight experimental groups: control (C-N, C-PA), antibiotics (AB-N, AB-PA), probiotics (P-N, P-PA), and antibiotics+probiotics (AB-P-N, AB-P-PA).
Early neurodevelopmental assessment was performed between postnatal days 7 and 9 using the righting reflex, negative geotaxis, and grip strength tests. The day following PA/normoxia, hippocampal tissue was collected for TNF-α quantification using ELISA.
Between postnatal days 45 and 65, offspring underwent late behavioral testing and were sacrificed on postnatal day 75. Brain tissue was harvested, fixed, paraffin-embedded, and processed for immunohistochemistry. Sections were immunostained for microglial (IBA-1) and astrocytic (GFAP) markers using a Mouse and Rabbit Specific HRP/DAB Detection IHC kit (ab64264) and analyzed using a Zeiss Axiolab 5 microscope.
Results
Offspring exposed to maternal antibiotics and/or PA exhibited impaired early neurodevelopmental reflexes and increased hippocampal inflammatory markers.
In the control group, exposure to PA induced marked activation of both microglia and astrocytes compared with normoxic conditions. IBA-1 immunostaining revealed a shift in microglial morphology from a ramified, surveillant phenotype to an amoeboid, activated state. GFAP immunolabeling demonstrated reactive astrocytosis in PA-exposed offspring.
Maternal antibiotic exposure induced microglial activation even under normoxic conditions, an effect further exacerbated by PA. Probiotic supplementation significantly attenuated antibiotic-induced microglial activation in both conditions.
Similarly, antibiotic-induced astrocytic activation was observed in normoxic offspring and intensified following PA. Probiotic administration mitigated astrocytic reactivity associated with both antibiotic exposure and perinatal asphyxia.
Conclusions
These findings indicate that probiotics modulate the microbiota–gut–brain axis, reduce neuroinflammation, and improve neurodevelopmental trajectories in offspring, supporting their potential as a preventive strategy against dysbiosis-related neurodevelopmental disturbances.