Late D-Chiro-Inositol Treatment Has Limited Effects on Hippocampal Alterations in Aged 3×Tg-AD Mice: Partial Rescue of CA1 Neurons Without Recovery of the Dentate Gyrus–CA3 Circuit

Background: Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder and the leading cause of dementia worldwide. Among the mechanisms implicated in AD progression, brain insulin resistance has been associated with impaired synaptic function and cognitive decline. In this context, inositols have emerged as promising neuroprotective compounds, and previous studies have reported beneficial effects of early D-chiro-inositol (DCI) administration in the 3×Tg mouse model. This exploratory study aimed to evaluate whether chronic oral DCI administration initiated at an advanced stage can modulate hippocampal neuronal density, synaptic markers and signaling pathways associated with insulin resistance in aged 3×Tg mice. Methods: Male and female wild-type and 3×Tg mice received DCI in drinking water (approximately 200 mg/kg/day) from 9 to 12 months of age. Neuronal density was assessed by unbiased counting of NeuN-positive cells (cells/mm2), and hippocampal protein expression was analyzed by Western blot, including markers related to insulin signaling, Tau phosphorylation, glutamatergic and endocannabinoid pathways, neuroinflammation and synaptic integrity. Sex effects were formally evaluated by three-way ANOVA (sex × genotype × treatment). Results: 3×Tg mice showed a lower neuronal density in the dentate gyrus–CA3 circuit, females displayed lower neuronal densities than males, and synaptophysin (males) and PSD95 (females) were reduced in 3×Tg mice. DCI increased neuronal density in the CA1 region of 3×Tg females but did not restore the neuronal deficit in the dentate gyrus–CA3 circuit or the synaptic markers. Compared with wild-type mice, 12-month-old 3×Tg animals displayed limited alterations in hippocampal signaling proteins, and DCI induced only sparse, often sex-dependent changes that neither normalized insulin signaling nor reduced Tau phosphorylation. Conclusions: Late-onset administration of DCI had limited effects on hippocampal alterations in aged 3×Tg mice. Although treatment ages were not directly compared, these findings contrast with previous results in which DCI treatment initiated at 3 months of age proved effective, and support the hypothesis that the therapeutic window for DCI narrows as pathology progresses. The sex-dependent differences observed further highlight the importance of considering sex as a biological variable in the design of therapeutic studies.