Introduction:

Alterations in gut microbiome composition have been associated with several neurodevelopmental and psychiatric disorders¹. In animal models, modifications of gut microbiome populations through dietary manipulation influence brain function and behavior, and have been shown to influence behavioral symptoms^2-6. With striking differences in microbiome-driven behavior, we employed diffusion tensor imaging (DTI) to explore whether these behavioral changes are also accompanied by corresponding changes in neural tissue microstructure.

Methods:

Male rats were assigned to one of four purified and irradiated diets: a control diet, a high-fat diet, a high-fiber diet, and a high-protein diet. Animals were fed the experimental diets immediately post-weaning for a total of 21 days. At post natal day (PND) 42, animals from each diet cohort (total n=20, n=5 per diet group, PRE) were sacrificed and brains dissected. The remaining animals (total n=24, n=6 per diet group, POST) were crossed over and remained on the control diet for an additional 21 days, whereupon the brains were isolated in a similar fashion. Brains were ex-vivo imaged and following standard preprocessing, tensors were reconstructed, registered, and normalized to a population-specific template. Tract-based spatial statistics (TBSS) were computed to evaluate whole-brain voxel-wise differences along standard diffusion tensor indices; permutation test results were considered significant at the α<0.05 level after family-wise error correction.

Results:

To uncover diet-induced changes in neural tissue microstructure, ex-vivo whole brain DTI was performed followed by a voxel-wise TBSS analysis on samples from both the PRE and POST diet groups. Figure 1 shows the TBSS experimental design; PRE diet group analysis (fig. 1a), POST diet group analysis (fig. 1b), and PRE-POST diet groups analysis (fig. 1c). Statistically significant differences in fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and trace (TR; trace: mean diffusivity [MD] x3) were identified. Figure 2 reveals substantial areas of differing FA, TR, and AD values for the high-fat diet group (B1:A1) principally concentrated in the neocortex and external capsule. Additionally, animals on the high-fiber diet (C1:A1) showed statistically significantly higher TR and AD in the hypothalamus, internal capsule, and brainstem, and statistically lower AD and RD values in the neocortex. Animals on the high-protein diet (D1:A1) demonstrated increased FA and RD in both the neocortex and cerebellum. TBSS analysis performed on POST animals also reveal widespread areas of increased FA, TR, AD, and RD in the high-fat group (B2:A2), which were found largely throughout the forebrain (fig. 3). Surprisingly, animals crossed over from the high-fiber diet (C2:A2) demonstrated tremendous changes in the diffusion tensor with widespread and global changes in the diffusion tensor with decreased FA, TR and AD, and increased RD that stand in stark contrast to the minimal changes see in the high-fiber PRE group (C1:A1). While no significant differences in FA and AD were identified in the POST high-protein group (D2:A2), statistically significant decreased TR and RD values were observed throughout the brainstem. Results for PRE-POST diet groups, shown in figure 4, reveal areas with differing FA and increased RD values in the neocortex and external capsule for the POST high-fat group (B2:B1). Significant TR, AD, and RD differences are showed in the case of high-fiber diet (C2:C1). Confluent TR and AD high values are present at the neocortex, external capsule, corpus callosum, and brainstem POST crossover. Increased RD is observed in the rest of the midbrain, inferior colliculus, and central gray. Lastly, the high-protein group (D2:D1) shows increased TR, AD, and RD values at the neocortex, external capsule, rest of the midbrain, and brainstem POST crossover; along with confluent increased TR and RD at the corpus callosum.

Summary & Discussion:

The mechanism through which the gut microbiome exerts its effects on the central nervous system (CNS) is multifactorial (neural, endocrine, and immunologic) but is thought to largely occur via the generation of bacterial metabolites, which exert their physiologic effects both locally and systemically. Short-chain fatty acids (SCFA), produced by the bacterial fermentation of dietary carbohydrates, alter neuronal excitability and gut bacteria also manufacture a wide spectrum of neuroactive compounds that include dopamine, γ-aminobutyric acid, histamine, acetylcholine and tryptophan, a precursor in the biosynthesis of serotonin^7,8. The results of our work extend and build upon these findings by uncovering changes in white matter structural integrity via differing DTI metrics that may be linked to diet-influenced gut microbiome populations. Our method for uncovering potential links between gut microbiome populations and brain structural changes can help guide important new experiments to study how these microbiome populations impact the CNS beyond transiently modulating the presence and flux of neuroactive molecules and compounds.

Acknowledgements

This work is supported under NIH awards UL1TR000427 and TL1TR000429. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.