Mechanical Properties Improve with Exercise and Environmental Complexity in Rat Model of Fetal Alcohol Spectrum Disorders Measured via MR Elastography

Introduction:: Fetal alcohol spectrum disorders (FASDs) can result from alcohol exposure (AE) during the prenatal period. Individuals diagnosed with FASDs suffer from impaired cognition, motor control, emotionality, and social behavior.¹ Neuroanatomical changes brought on by AE include abnormal myelination and white matter connectivity, particularly in executive function-related neural circuitry.² Behavioral treatments for AE are nonexistent, but exercise has been shown to enhance proliferation of neuronal precursors and increase white matter growth.^3,4 Furthermore, environmental complexity following exercise extends the survival of newly generated cells in rats.⁵ Imaging methods sensitive to brain tissue microstructure can be used to assess brain integrity in FASD and monitor response to therapy. Magnetic resonance elastography (MRE) is a noninvasive, quantitative MRI technique used to estimate mechanical properties. While preclinical brain MRE research is limited,^6,7 we validated a technique for rodent MRE using the nonlinear inversion algorithm (NLI) with translatable quality and resolution to humans.⁸ In this longitudinal study, we investigate the effects of AE on mechanical properties in the developing rodent brain. Additionally, we test whether behavioral “superintervention,” consisting of combined exposure to wheel running (WR) followed by environmental complexity (EC), can mitigate AE-induced changes to MRE measures.


Materials and Methods:: To model FASD, female Long Evans rat pups (n=9) were given alcohol (5.25 g/kg) in milk formula twice per day from postnatal days (PD) 4-9 via intragastric intubation. Sham intubated (SI) controls were not administered any liquid during intubation (n=11). All rats were weaned and socially housed at PD23.

Between PD25-29, MRE scans were performed on animals anesthetized with 1-3% isoflurane using a Bruker 9.4T preclinical imaging system. For the MRE scans, we used a nonmagnetic, piezoelectric actuator and bite bar driver to apply vibrations at 800 Hz. A resolution of 0.25x0.25x0.5 mm³ was achieved in 32 minutes using a custom MRE-EPI sequence.

Following the first scan (TP1), half of the rats were assigned to the wheel running/environmental complexity (WR/EC) superintervention group. They were pair-housed in cages with free access to an attached running wheel where they could exercise for 12 days (PD30-42). WR/EC rats were then transferred to environmental complexity cages (6-9 animals/cage). These cages were furnished with various objects and toys that were changed every other day. The remaining rats were returned to social housing (SH). After 30 days, all rats received their second MRE scan (TP2). Figure 1 shows the timeline of this study.

Mechanical properties were estimated from MRE displacement data using isotropic NLI with a no boundary condition formulation (NoBC-NLI).⁹ NLI outputs storage modulus (G’) and loss modulus (G’’) which were used to calculate shear stiffness (μ).


Results, Conclusions, and Discussions:: Figure 2 shows AE rats exhibited softer brain tissue than SI controls at TP1 (SI = 6.59 kPa, AE = 5.94 kPa, p = 0.012); this stiffness difference likely reflects lower white matter integrity in AE rats. While the AE rats had slightly lower stiffness than SI rats at TP2 (SI = 6.84 kPa, AE = 6.46 kPa, p = 0.069), this difference was less than at TP1, suggesting that the superintervention led to stiffness recovery. Indeed, the longitudinal analysis (Figure 3) indicated there was no difference from TP1 to TP2 for SI (Δμ = 0.26 kPa, p = 0.32), though the AE group saw a significant increase in stiffness over time (Δμ = 0.52 kPa, p = 0.020). The WR/EC condition also had a positive effect on stiffness (Δμ = 0.45 kPa, p = 0.010) which agrees with the hypothesis that superintervention proliferates neuronal growth; there was also no significant difference for the SH control condition (Δμ = 0.28 kPa, p = 0.39). This conclusion was reinforced by a significant increase in stiffness only within the AE/WR/EC subgroup (Δμ = 0.62 kPa, p = 0.029).

This study demonstrates that exercise followed by environmental complexity has a positive effect on rodent brain mechanical properties. The superintervention particularly improved the stiffness of alcohol exposed rats over time, such that brain stiffness recovered to near baseline values. Furthermore, we confirmed MRE is sensitive to small property variations in rodents which may reflect myelination and microstructural integrity. In future work, we will correlate these results with immunohistochemistry and investigate how fetal alcohol exposure and superintervention impact anisotropic properties in white matter throughout development.


Acknowledgements (Optional): : NIH/NIBIB R01 EB027577 (Johnson)
NIH/NIAAA R01AA027269-01 (Klintsova)
NIH 2P20GM10365 (University of Delaware Center for Biomedical and Brain Imaging; Klintsova and Johnson)


References (Optional): :

1. Streissguth, A. P., Aase, J. M., Clarren, S. K., Randles, S. P., LaDue, R. A., and Smith, D. F. (1991). Fetal alcohol syndrome in adolescents and adults. JAMA J. Am. Med. Assoc. 266, 1077.


2. Wilhelm, C. J., and Guizzetti, M. (2016). Fetal alcohol spectrum disorders: An overview from the glia perspective. Front. Integr. Neurosci. 9, 1–16.


3. Helfer, J. L., Goodlett, C. R., Greenough, W. T., and Klintsova, A. Y. (2009). The effects of exercise on adolescent hippocampal neurogenesis in a rat model of binge alcohol exposure during the brain growth spurt. Brain Res. 1294, 1–11.


4. Milbocker, K. A., LeBlanc, G. L., Brengel, E. K., Hekmatyar, K. S., Kulkarni, P., Ferris, C. F., et al. (2022). Reduced and delayed myelination and volume of corpus callosum in an animal model of Fetal Alcohol Spectrum Disorders partially benefit from voluntary exercise. Sci. Rep. 12, 1–17.


5. Hamilton, G. F., Boschen, K. E., Goodlett, C. R., Greenough, W. T., and Klintsova, A. Y. (2012). Housing in Environmental Complexity Following Wheel Running Augments Survival of Newly Generated Hippocampal Neurons in a Rat Model of Binge Alcohol Exposure During the Third Trimester Equivalent. Alcohol. Clin. Exp. Res. 36, 1196–1204.


6. Bayly P V., Garbow JR. Pre-clinical MR elastography: principles, techniques, and applications. J Magn Reson. 2018;291:73-83.


7. Bigot M, Chauveau F, Beuf O, Lambert SA. Magnetic resonance elastography of rodent brain. Front Neurol. 2018;9(NOV):1-8.


8. Williams LT, Milbocker KA, Sullivan SR, et al. Mapping Rodent Brain Mechanical Properties In Vivo with Magnetic Resonance Elastography and Nonlinear Inversion. In: Proc. Intl. Soc. Mag. Reson. Med. 2022:10-12.


9. McGarry MDJ, Van Houten EEW, Johnson CL, et al. Multiresolution MR elastography using nonlinear inversion. Med Phys. 2012;39(10):6388-6396.