Article Figures & Data
Figures
- Figure 1.
Blood ketone levels and stability 1 week after administration of ketogenic diet in uninjured mice. A, Blood samples were collected from the tail vein and determined by ketone testing strips and monitoring. Significantly higher ketone blood concentrations were found in mice fed the ketogenic diet compared with a conventional pellet diet. N = 10 each for KD and PD; ****p < 0.0001. B, Mice fed a ketogenic diet reliably remained in ketosis with KD being introduced 7 d prior. In addition, within 1 h of being given a pellet diet, mice were no longer in a state of ketosis. N = 2 for KD; N = 3 for PD. C, Comparison of body weights between mice who were fed the ketogenic and pellet diets without surgical intervention; no significant difference was detected at each time point between the diets. N = 6–10 each for PD and KD. Error bars indicate the mean ± SEM.
- Figure 2.
The effect of diets on neuromodulator content in the lumbar spinal cord of mice. A, B, Mice fed the ketogenic diet had significantly lower content of the monoamine precursors tyrosine (A) and tryptophan (B); **p = 0.0041 and *p = 0.0209, respectively. C–E, No changes were detected in the levels of monoamines noradrenaline (C), dopamine (D), and serotonin (5-HT; E). There were also no changes in the content of the main metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA; F). N = 18 for PD; N = 6 for KD. Error bars indicate the mean ± SEM.
- Figure 3.
The effect of diet on open-field locomotor behaviors in a model of peripheral nerve injury. Peripheral nerve injury was modeled by SNI; comparison of general locomotor behaviors were made at before (baseline) and after SNI. A–F, No significant differences were observed between the diets in distance traveled (A, B), duration of in-place activity (C, D), and number of crossings from the periphery of the open field box to the center (E, F). G, At baseline, mice fed the ketogenic diet displayed more in-place activity in the center of the box. *p = 0.0138. H, However, no effects of diet were observed after SNI between mice fed a pellet or ketogenic diet. Duration of locomotion in the center of the open field box is a proxy measure of anxiety-like behaviors. The more time spent in the center of the box suggests less anxiety as mice innately avoid bright open spaces. I, At baseline, no significant movement to the center was observed; p = 0.054. J, No effects of the diet were observed after SNI. N = 7–8 for SHAM; N = 8 for SNI + PD; N = 8 for SNI + KD. Error bars indicate the mean ± SEM.
- Figure 4.
The effect of diet on other sensorimotor behaviors before and after peripheral nerve injury. Comparison of mechanical withdrawal threshold (von Frey hair test) and fine motor behaviors (ladder rung) made before (baseline) and after SNI. A, No difference in the mechanical withdrawal threshold between the diets at baseline. B, Mechanical withdrawal threshold was significantly lower in SNI mice compared with sham mice, indicating the development of mechanical allodynia (pain evoked by normally innocuous stimulations). Post hoc Bonferroni analysis revealed a significant difference between the SHAM+PD and SNI+KD groups on days 3 and 7 after SNI; %p = 0.0038, #p = 0.0117, respectively. The SNI+PD group also had a lower mechanical withdrawal threshold compared with SHAM+PD on day 7 after SNI, $p = 0.0428. Three days after SNI, the SNI+KD group also had significantly lower mechanical withdrawal threshold compared with the SNI+PD group; *p = 0.0231. C, During Ladder rung, there was no difference in the step score of animals at baseline between animals fed the KD vs PD. D, Over the course of 28 days recovery, diet produced no significant changes in recovery. N = 7 for SHAM group, N = 8 for SNI+KD and SNI+PD groups. Error bars indicate the mean ± SEM.
- Figure 5.
The effect of diet on open-field locomotor behaviors in a model of spinal cord injury. SCI was modeled by a hemisection at T12–T13, on the left side of the spinal cord. A comparison of general locomotor behaviors was made before (baseline) and after SCI. A, B, Distance traveled after 30 min in the OFT; there was no significant difference at baseline between PD-fed and KD-fed animals (A) or following injury (B). C, D, No difference in the number of crosses from the periphery of the open field box to the center. E, G, At baseline, mice fed the ketogenic diet displayed no differences in activity in place in the entirety of the arena (E) or in the center of the box (G). F, H, No significant effects of diet were observed for in-place activity after SCI between PD- and KD-fed mice in either the arena (F) or the center of the box (H). The duration of locomotion in the center of the open field box is a proxy measure of anxiety-like behaviors. The more time spent in the center of the box indicates less anxiety as mice innately avoid bright open spaces. I, Nonsignificant difference at baseline in mice fed a KD versus mice fed a PD during locomotion in the center of the open field (Student’s t test, p = 0.548). J, No effects of the diet were observed after SCI except for in the time domain; two-way ANOVA, p = 0.008. There were no significant differences between animals in the KD group and the PD group following injury (two-way ANOVA, p = 0.8994). N = 8 for SHAM, N = 8 for the SCI+PD group, N = 8 for SCI + KD group. Error bars indicate the mean ± SEM.
- Figure 6.
The effect of diet on other sensorimotor behaviors before and after spinal cord injury. SCI was modeled by a hemisection T12–T13, left side of the spinal cord. Comparison of mechanical withdrawal threshold (von Frey hair test) and fine motor behaviors (ladder rung) was made at before (baseline) and after SNI. A, B, No difference in mechanical withdrawal threshold between the diets at baseline (A) or after SCI (B). C, There was no difference in the step score of animals at baseline between animals fed the KD versus those fed the PD. D, Diet produced no significant changes over the course of 28 d on the rehabilitation of motor recovery. N = 8 each for SHAM, SNI+KD, and SNI+PD. Error bars indicate the mean ± SEM.
Tables
Macronutrients PD (LabDietmousediet 20) KD (Bio-ServF3666) Measurement Protein 24.65% 8.60% % Fat 13.21% 75.10% % Carbohydrate 62.14% 3.20% % Other Fiber 4.00% 4.80% % Ash 6.10% 3.00% % Moisture 12.00% <10% % Amino acids Alanine 1.15% 0.23% % Arginine 1.22% 0.31% % Aspartic Acid 2.19% 0.55% % Cystine 0.28% 0.03% % Glutamic acid 4.34% 1.73% % Glycine 0.96% 0.21% % Histidine 0.5% 0.23% % Isoleucine 0.97% 0.47% % Leucine 1.56% 0.71% % Lysine 1.16% 0.63% % Methionine 0.7% 0.22% % Phenylalanine 0.9% 0.38% % Proline 1.47% 0.87% % Serine 1.03% 0.48% % Threonine 0.77% 0.37% % Tryptophan 0.26% 0.1% % Tyrosine 0.59% 0.48% % Valine 1% 0.55% % Minerals Calcium 0.81% 0.57% % Chloride 0.42% 0.17% % Copper 13 ppm 6.6 ppm ppm Chromium 0.81 ppm 2.2 ppm ppm Fluoride/fluorine 10 ppm 0.0 ppm ppm Iodine 1.5 ppm 0.2 ppm ppm Iron 220 ppm 38.7 ppm ppm Magnesium 0.16% 0.56% % Manganese 85 ppm 63.6 ppm ppm Phosphorus 0.6% 0.49% % Potassium 0.7% 0.39% % Selenium 0.3 ppm 0.19 ppm ppm Sodium 3000 ppm 1128 ppm ppm Sulfur 3400 ppm 366 ppm ppm Zinc 87 ppm 36 ppm ppm Comparison table of the two diets used throughout this study. All values have been converted to the percentage based on weight from company data sheets of the diets. PD Pico-Vac Mouse Diet 20 from LabDiet compared with KD #F3666 6:1 from Bio-Serv. Diets have been sorted into four categories for readability: Macronutrients, Other, Amino acids, Minerals.
Score Metz scale2009 Explanation 0 Total miss 0 points were assigned when the limb completely missed a rung (i.e. it did not touch it), and a fall occurred. Afall was defined as a limb deeply falling in between rungs with body posture and balance being disturbed. 1 Deep slip The limb was initially placed on a rung, then slipped off as the mouse began to bear weight, causing theanimal to fall. 2 Slight slip The limb was placed on a rung, then slipped off as the mouse began to bear weight, but it did not resultin a fall or interrupt the step cycle. In this case, the animal was able to maintain balance and continuea coordinated gait. 3 Replacement The limb was placed on a rung, but before it was bearing weight it was quickly lifted and placed onanother rung. 4 Correction The limb was aimed for one rung but was then placed on another rung without touching the first one.Alternatively, a score of 4 was recorded if a limb was placed on a rung and was quickly repositionedwhile remaining on the same rung. 5 Partialplacement The limb was placed on a rung with either wrist or digits of the forelimb or heel or toes of the hindlimb. 6 Correctplacement The midplantar surface of a limb was placed on the rung with full weight support. This 6 point scale was used to score the mouse hindlimb positioning when crossing over the ladder-run test, 0 being the complete miss of the hindpaw placement on the ladder rung, up to a maximum score of six with correct paw placement achieved. This scale was used throughout the experiments and was adapted from Metz and Whishaw (2009).
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