Microglia-associated Galectin-3 expression in prenatal stress-induced cognitive dysfunction in adult offspring

To characterize Galectin-3 expression and its cellular distribution in the hippocampus and prefrontal cortex of adult offspring exposed to prenatal stress, and to examine their associations with microglial activation, neuroinflammation, and cognitive dysfunction. Twelve pregnant Sprague–Dawley rats were randomly assigned to a stress group ( n = 6) or a control group ( n = 6). Rats in the stress group were subjected to restraint stress from gestational day 15 to day 21, whereas rats in the control group received no intervention. Cognitive function in adult offspring was assessed using the open-field test, elevated plus maze test, novel object recognition test, and Y-maze test. An enzyme-linked immunosorbent assay (ELISA)was used to measure peripheral blood levels of corticotropin-releasing hormone (CRH) and corticosterone (CORT) in dams and offspring. ELISA was used to measure the expression levels of inflammatory cytokines in the serum of offspring, while quantitative real-time PCR (qRT-PCR) was applied to detect the mRNA expression levels of inflammatory cytokines in the hippocampus, and prefrontal cortex of offspring. Immunofluorescence staining was used to evaluate microglial activation by assessing Iba1-positive microglial morphology and quantifying Iba1/CD68 double-positive cells, as well as to examine the colocalization of Iba1 with Galectin-3 in the hippocampus and prefrontal cortex of offspring. qRT-PCR was used to determine Galectin-3 mRNA expression levels in the hippocampus and prefrontal cortex of offspring. Western blotting was performed to assess the protein expression levels of Galectin-3, NF-κB p65, and the neuronal marker NeuN in the hippocampus and prefrontal cortex of offspring. Compared with the control group, offspring in the prenatal stress group exhibited significant behavioral impairments in the open-field test, elevated plus maze test, novel object recognition test, and Y-maze test. ELISA results showed that prenatal stress increased peripheral blood levels of CRH and CORT in both dams ( P < 0.05) and offspring ( P < 0.001). ELISA and qRT-PCR analyses further revealed significantly increased expression of interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in the peripheral blood, hippocampus, and prefrontal cortex of offspring ( P < 0.05). Immunofluorescence staining demonstrated that prenatal stress induced activation-associated morphological changes in Iba1-positive microglia and significantly increased the numbers of Iba1/CD68 double-positive cells in the hippocampus and prefrontal cortex (all P < 0.01). The numbers of Iba1/Galectin-3 double-positive cells were also significantly increased in the hippocampus ( P < 0.05) and prefrontal cortex ( P < 0.001). qRT-PCR and Western blot analyses showed that prenatal stress significantly upregulated Galectin-3 expression in the hippocampus and prefrontal cortex of offspring and increased total NF-κB p65 protein expression, accompanied by decreased NeuN expression (all P < 0.05). Increased Gal-3 expression was associated with microglial neuroinflammatory responses and cognitive dysfunction in adult offspring exposed to prenatal stress.