Dynamic Influence of the Estrous Cycle on Hippocampal Stiffness: Insights from Magnetic Resonance Elastography of the Female Rat Brain

Introduction: Sex hormone fluctuations across the estrous/menstrual cycle temporally affect female brain microstructure. Variation in sex hormone levels during the estrous cycle in adult female rats alters dendritic spine density on hippocampal CA1 pyramidal neurons (Woolley 1990), as estrogen induces spinogenesis and both estrogen and progesterone increase synaptogenesis in the hippocampus (Hara 2015, Pletzer 2023). Traditional neuroimaging techniques have captured the influence of sex hormones, particularly on the volume of the CA1 subregion, in both human and rodent subjects (Zsido 2023, Qui 2013), but volumetric measures do not provide information on tissue microstructure.

Magnetic resonance elastography (MRE) is a noninvasive neuroimaging technique that can measure mechanical properties (i.e. stiffness and damping ratio) in specific subregions of the brain by imaging small tissue deformations and estimating material parameters. MRE produces spatially-resolved maps of brain tissue mechanical properties via inverse solution of the underlying equations of motion. Studies have used MRE to characterize neurological disease and quantify normal tissue properties (Murphy 2019, Hiscox 2021), but sex differences in mechanical properties and factors particularly affecting the female brain are understudied.

This longitudinal study uses a preclinical rat brain MRE protocol (Milbocker 2017) to evaluate changes in hippocampal microstructure across the estrous cycle. Rats experience rapid fluctuations in sex hormones due to their 4-day estrous cycle, indicating dynamic changes in brain microstructure occur on a daily basis. These findings in addition to histological data aim to bridge existing gaps in our understanding of how brain mechanics and mechanobiology are impacted by the neuroendocrine system.

Materials and

Methods: Adult female Long-Evans rats (n=8) with regular 4-day estrous cycles were estrous tested via vaginal swabbing to confirm phase, then sequentially MRE scanned during each of the 4 phases (proestrus, estrus, metestrus, and diestrus). MRE data were acquired with a custom echoplanar imaging sequence with a 9.4T Bruker Biospec MRI scanner. A piezoelectric actuator was attached to a custom bite bar to deliver vibrations at 700 Hz to anesthetized rats maintained on 1-3% isoflurane in oxygen and at 32-36℃ for a total 1-hour scan protocol. MRE imaging parameters included: TE/TR = 60/4000 ms; 20x20 mm FOV; 80x80 matrix; 40 slices, 0.5 mm thick; 0.25x0.25x0.50 mm resolution. Images with OSS-SNR < 3 were discarded and the rat was rescanned at the same phase (McGarry 2011). Maps of mechanical properties, including stiffness and damping ratio (Figure 1), were estimated from displacement images with nonlinear inversion (NLI) formulated without boundary conditions (McGarry 2012, Kurtz 2023). Brain regions were manually masked, and regional mechanical properties were normalized using whole brain values.

Results, Conclusions, and Discussions: We performed within-rat, repeated measures ANOVA on the longitudinal stiffness and damping ratio values from the whole brain, the hippocampus, and the hippocampal CA1 subregion between each cycle phase. As expected, the whole brain showed no significant changes to stiffness or damping ratio between phases, though both parameters were affected in whole hippocampus and CA1 subregion. Hippocampal stiffness showed no significant change between proestrus and estrus, increased from estrus to metestrus (p < 0.001), decreased from metestrus to diestrus (p=0.037), and decreased from diestrus to proestrus (p < 0.001; see Figure 2A). CA1 stiffness increased from proestrus to estrus (p=0.007), increased from estrus to metestrus (p < 0.001), and decreased from metestrus to diestrus (p < 0.001; see Figure 2C). CA1 damping ratio decreased from estrus to metestrus (p=0.012) and increased from metestrus to diestrus (p=0.038; see Figure 2D).

We have demonstrated that the estrous phase exerts a notable influence on hippocampal tissue mechanical properties in the female rat brain. Initially, we expected a decline in stiffness from proestrus to estrus because of the documented reduction in dendritic spine density that results from simultaneous depletion of estrogen and progesterone occurring between these phases. However, we observed an increase in stiffness in estrus, persisting into metestrus, despite a reduction in dendritic spines. This finding is likely attributable to the reduction in newly formed, immature dendritic spines, which are extremely “soft” compared to their mature counterparts (Smith 2007). The continued increase in CA1 stiffness in metestrus suggests that the cytoarchitecture stabilizes before estrogen and progesterone peak again in the diestrus-proestrus transition.

Neurons are highly mechanoresponsive, therefore the changing mechanical properties across the cycle may influence neuronal cell function. The next phase of this research is to manipulate hormone fluctuations in the female rat to identify unique contributions of estrogen and progesterone to brain tissue mechanics. Ultimately, this work is fundamental for understanding the unique changes to brain tissue mechanics in neurological diseases that are highly prevalent in females.

Acknowledgements (Optional): This research was supported in part by NIH grant R01-EB027577, the Delaware CTR Program (U54-GM104941), and the Delaware Neuroscience CORBE (P20-GM103653).