Abstract
Repetitive mild traumatic brain injury (rmTBI) is associated with a range of neural changes which is characterized by axonal injury and neuroinflammation. Ketogenic diet (KD) is regarded as a potential therapy for facilitating recovery after moderate-severe traumatic brain injury (TBI). However, its effect on rmTBI has not been fully studied. In this study, we evaluated the anti-neuroinflammation effects of KD after rmTBI in adolescent mice and explored the potential mechanisms. Experimentally, specific pathogen-free (SPF) adolescent male C57BL/6 mice received a sham surgery or repetitive mild controlled cortical impacts consecutively for 7 days. The uninjured mice received the standard diet, and the mice with rmTBI were fed either the standard diet or KD for 7 days. One week later, all mice were subjected to behavioral tests and experimental analysis. Results suggest that KD significantly increased blood beta-hydroxybutyrate (β-HB) levels and improved neurological function. KD also reduced white matter damage, microgliosis, and astrogliosis induced by rmTBI. Aryl hydrocarbon receptor (AHR) signaling pathway, which was mediated by indole-3-acetic acid (3-IAA) from Lactobacillus reuteri (L. reuteri) in gut and activated in microglia and astrocytes after rmTBI, was inhibited by KD. The expression level of the toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88) in inflammatory cells, which mediates the NF-κB pathway, was also attenuated by KD. Taken together, our results indicated that KD can promote recovery following rmTBI in adolescent mice. KD may modulate neuroinflammation by altering L. reuteri in gut and its metabolites. The inhibition of indole/AHR pathway and the downregulation of TLR4/MyD88 may play a role in the beneficial effect of KD against neuroinflammation in rmTBI mice.
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The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.
Abbreviations
- 3-IAA:
-
Indole-3-acetic-acid
- AHR:
-
Aryl hydrocarbon receptor
- β-HB:
-
Beta-hydroxybutyrate
- CXCL1:
-
Chemokine (C-X-C motif) ligand 1
- CCI:
-
Controlled cortical impact
- DTI:
-
Diffusion tensor imaging
- ELISA:
-
Enzyme-linked immunosorbent assay
- FA:
-
Fractional anisotropy
- GFAP:
-
Glial fibrillary acidic protein
- HE:
-
Hematoxylin and eosin
- IL-1β:
-
Interleukin β1
- IRF-3:
-
Interferon regulatory Factor 3
- Iba-1:
-
Ionized calcium-binding adapter molecule 1
- KD:
-
Ketogenic diet
- MyD88:
-
Myeloid differentiation primary response 88
- MCT1:
-
Monocarboxylate transporter 1
- MD:
-
Mean diffusivity
- NF-κB:
-
Nuclear factor kappa light chain enhancer of activated B cells
- OCT:
-
Optimal cutting temperature compound
- PBS:
-
Phosphate-buffered saline
- rmTBI:
-
Repetitive mild traumatic brain injury
- TLR4:
-
Toll-like receptor 4
- TNF:
-
Tumor necrosis factor
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Acknowledgements
We thank the entire the laboratory of Neurosurgery Department for supporting experimental space and technical services.
Funding
This work was supported by grants from the National Natural Science Foundation of China (No. 82071359), the Shanghai Rising-Star Program (21QA1405600), the Natural Science Foundation of Shanghai (21ZR1439000) and Ren ji New Star Program (F. Jia).
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DD, FZ and YJ designed the experiments. DD, FZ and SS performed and analyzed most experiments with the help of TL, MC, QL. DD and FZ were major contributors in writing this manuscript. FJ and XZ participated in the discussions and revised the manuscript. YJ supervised the entire project and was responsible for finalizing and submitting the manuscript. All authors read and approved the final manuscript.
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Animal protocols were approved by the Animal Care and Experimental Committee of the School of Medicine of Shanghai Jiao Tong University.
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10571_2022_1226_MOESM3_ESM.tif
Supplementary Figure 1 Serum levels of total bilirubin, biliverdin and hemin in mice of different groups. Bilirubin: n = 6; one-way ANOVA; *p = 0.0211 SCG vs TCG, *p = 0.0489 SCG vs TKG, p = 0.9017 TCG vs TKG; F2, 15 = 0.2121, p = 0.8113; biliverdin: n = 6; one-way ANOVA; p = 0.2009 SCG vs TCG, p = 0.0710 SCG vs TKG, p = 0.8210 TCG vs TKG; F2, 15 = 0.08621, p = 0.9179; hemin: n = 6; one-way ANOVA; p = 0.3920 SCG vs TCG, p = 0.5662 SCG vs TKG, p = 0.9483 TCG vs TKG; F2, 15 = 0.01908, p = 0.9811. Supplementary file3 (TIF 4196 KB)
10571_2022_1226_MOESM4_ESM.tif
Supplementary Figure 2 The morphological changes of microglia and astrocytes. A, C Fluorescence images showing microglia and astrocytes in the hippocampal CA1 region. Scale bar = 10 μm.B Graphs showing the morphological analyses in Iba-1+ microglia. Soma area: n = 6; one-way ANOVA; p = 0.2099 SCG vs TCG, p = 0.9224 SCG vs TKG, p = 0.1106 TCG vs TKG; F2, 15 = 1.281, p = 0.3065; process length: n = 6; one-way ANOVA; *p = 0.0102 SCG vs TCG, p = 0.7394 SCG vs TKG, *p = 0.0444 TCG vs TKG; F2, 15 = 1.452, p = 0.2653.D Graphs showing the morphological analyses in GFAP+ astrocytes. Soma area: n = 6; one-way ANOVA; p = 0.3629 SCG vs TCG, p = 0.2174 SCG vs TKG, *p = 0.0167 TCG vs TKG; F2, 15 = 0.09059, p = 0.9139; process length: n = 6; one-way ANOVA; *p = 0.0152 SCG vs TCG, p = 0.9847 SCG vs TKG, *p = 0.0212 TCG vs TKG; F2, 15 = 0.2645, p = 0.7711. Supplementary file4 (TIF 6632 KB)
10571_2022_1226_MOESM5_ESM.tif
Supplementary Figure 3 Negative control immunofluorescence images of AHR/RelB colocalization in microglia from the hippocampal CA1 region. Scale bar = 20 μm. Supplementary file5 (TIF 13068 KB)
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Dilimulati, D., Zhang, F., Shao, S. et al. Ketogenic Diet Modulates Neuroinflammation via Metabolites from Lactobacillus reuteri After Repetitive Mild Traumatic Brain Injury in Adolescent Mice. Cell Mol Neurobiol 43, 907–923 (2023). https://doi.org/10.1007/s10571-022-01226-3
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DOI: https://doi.org/10.1007/s10571-022-01226-3