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. 2024 Aug 31;17(9):1160.
doi: 10.3390/ph17091160.

Neuroregeneration Improved by Sodium-D,L-Beta-Hydroxybutyrate in Primary Neuronal Cultures

Csilla Ari  1   2 , Dominic P D'Agostino  2   3   4 , Byeong J Cha  3
Affiliations

Neuroregeneration Improved by Sodium-D,L-Beta-Hydroxybutyrate in Primary Neuronal Cultures

Csilla Ari et al. Pharmaceuticals (Basel). .

Abstract

Ketone bodies are considered alternative fuels for the brain when glucose availability is limited. To determine the neuroregenerative potential of D,L-sodium-beta-hydroxybutyrate (D/L-BHB), Sprague Dawley rat primary cortical neurons were exposed to simulated central nervous system injury using a scratch assay. The neuronal cell migration, cell density and degree of regeneration in the damaged areas (gaps) in the absence (control) and presence of BHB (2 mM) were documented with automated live-cell imaging by the CytoSMART system over 24 h, which was followed by immunocytochemistry, labeling synapsin-I and β3-tubulin. The cell density was significantly higher in the gaps with BHB treatment after 24 h compared to the control. In the control, only 1.5% of the measured gap areas became narrower over 24 h, while in the BHB-treated samples 49.23% of the measured gap areas became narrower over 24 h. In the control, the gap expanded by 63.81% post-injury, while the gap size decreased by 10.83% in response to BHB treatment, compared to the baseline. The cell density increased by 97.27% and the gap size was reduced by 74.64% in response to BHB, compared to the control. The distance travelled and velocity of migrating cells were significantly higher with BHB treatment, while more synapsin-I and β3-tubulin were found in the BHB-treated samples after 24 h, compared to the control. The results demonstrate that D/L-BHB enhanced neuronal migration and molecular processes associated with neural regeneration and axonogenesis. These results may have clinical therapeutic applications in the future for nervous system injuries, such as for stroke, concussion and TBI patients.

Keywords: BHB; beta-hydroxybutyrate; cell migration; exogenous ketones; ketone salt; neural injury; neuroplasticity; scratch assay; synapsin; tubulin.

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Conflict of interest statement

International Patent # PCT/US2014/031237, University of South Florida, D.P.D’Agostino, S. Kesl, Patrick Arnold, “Compositions and Methods for Producing Elevated and Sustained Ketosis”. Patent: US 10,980,764 B1, C. Ari, D.P.D’Agostino, “Exogenous ketone supplements for reducing anxiety-related behavior”; Ari, C., D’Agostino, D.P. Technology Title: “Exogenous Ketone Supplementation Improved Motor Function in Sprague–Dawley Rats”. USF Ref. No: 16A019; Ari, C., D’Agostino, D.P. Technology Title: “Lowering of Blood Glucose in Exercising and Non-Exercising Rats Following Administration of Exogenous Ketones and Ketone Formulas”. USF Ref. No: 16A049; Ari, C., D’Agostino, D.P. Technology Title: “Neuroregeneration improved by ketone”. USF Ref. No: 16B128 (provisional patent); Patent: US 10,945,975 B2: Ari, C., D’Agostino, D.P. Dean, J.B. Technology Title: “Delaying latency to seizure by combinations of ketone supplements”. Non provisional patent No. 210112–9018-US02 for AC and DPD. Technology Title: “Methods of Increasing Latency of Anesthetic Induction Using Ketone Supplementation”. US Patent Application No. 17/576,375, patent: Z. Kovacs, C. Ari, D.P.D’Agostino: “Ketone supplements evoked effect on absence epilepsy by co-administration of Uridine”, D.P. D’Agostino and C. Ari are the co-owners of the company Ketone Technologies LLC, and C. Ari is the owner of Fortis World LLC. These interests have been reviewed and managed by the University in accordance with its Institutional and Individual Conflict of Interest policies. All authors declare that there are no additional conflicts of interest.

Figures

Figure 1
Figure 1
In response to D/L-BHB treatment, there was a significant increase in cell density in the damaged areas after 24 h, compared to the baseline (p = 0.001) and compared to the control (p = 0.0053); (A). The gap size significantly decreased in response to BHB treatment, compared to the baseline (p = 0.0019), while the gap size increased in the control (p < 0.0001) 24 h after the simulated injury (B). ** p < 0.01, **** p < 0.0001.
Figure 2
Figure 2
There was a significant increase in cell density in response to D/L-BHB treatment, compared to the control, in the damaged areas 24 h after simulated injury. (A) The control group after 24 h: fewer cells were found in the damaged area/gap, compared to the BHB-treated cells. (B) The BHB-treated group after 24 h: more cells were found in the damaged area, compared to the control, and the gap of the damaged area almost closed at some areas (arrows). (C) The control and (D) BHB-treated cells at baseline/at time of injury and (E) the control and (F) BHB-treated cells 24 h after injury.
Figure 3
Figure 3
DAPI staining shows the cell density in the damaged areas 24 h after injury. (A–C) In the control, a lower density of cell nuclei was found in the damaged areas; (D–F) With the BHB treatment, more DAPI-stained cell nuclei were found in the damaged areas, compared to the control.
Figure 4
Figure 4
Multiple parameters improved in D/L-BHB-treated samples 24 h after injury. (A) The velocity of the cells migrating in the BHB-treated sample was significantly higher (p = 0.00041), compared to the control. The graph shows individual values as well as the mean. (B) The estimated plot of the distance traveled by cells migrating in the BHB-treated samples were higher, compared to the control (p = 0.0041). (C) Significantly more synapsin-I was found in the BHB-treated samples, compared to the control (p = 0.006). (D) Significantly more beta-III-tubulin was found in the BHB-treated samples, compared to the control (p = 0.0435). * p < 0.05, ** p < 0.01.
Figure 5
Figure 5
Immunofluorescent staining shows that the density of cell nuclei (blue), synapsin-I (red) and beta-III-tubulin (green) increased with D/L-BHB treatment 24 h after injury. (A,B) In the control, fewer cell nuclei were visible around the regeneration site, and there was a lower density of synapsin-I and beta-III-tubulin. (C,D) With the BHB treatment, more cell nuclei were visible around the regeneration site, and there was a higher density of synapsin-I and beta-III-tubulin, compared to the control. Scale bar is 100 μm (A,C); 50 μm (B,D).
Figure 6
Figure 6
Summary of potential mechanisms that may contribute to the effect of D/L-BHB on neuroregeneration.
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