Ketogenic diet treatment (3:1 KD) is neuroprotective after C5 hemi-contusion (experiment 1).

<p>(A-B) Representative pictures of emerald traced corticospinal tract (CST) axons 14 wks post-injury (Zeiss Axioplan fluorescent microscope). A higher magnification of the box in figure A and B is shown in respectively A’ and B’, scale bar: 500 μm. (A’) Grey matter and (B’) CST sparing measurements, scale bar: 500 μm. Solid line I: length grey matter, sparing dashed line II: length ipsilateral CST sparing. (C) The lesion area at the epicenter and (D) spared grey tissue was significantly increased in spinal cords of KD fed animals compared to the standard diet group (SD). Error bars indicate SEM. * p=0.026, # p=0.030 (t-test, one tailed).</p

Glucose transporter-1 (GLUT1) immunoreactivity increased in the capillaries of the lesion penumbra in rats on ketogenic diet.

<p>(A) Representative pictures of spinal cord section showing GLUT1 staining characteristic of blood vessels at 7 days following injury (5x magnification; Leica DM5000B microscope, Leica Microsystems). Note the enhanced GLUT1 staining at the penumbra of the lesion cavity. To count number of Glut1 positive capillaries a 750×750 μm window was placed over the gray matter, with bottom of the window always touching boundary between gray and white matter (represented by box I). Scale bar: 500 μm (B) Higher magnification of representative field for GLUT1 quantification analysis of the SD and KD group, scale bar: 100 μm. (C) Blood vessel density, as measured by GLUT1 staining, increased by ~30% in the KD group compared to the SD group. Error bars indicate SEM. * p ≤ 0.05 (t-test).</p

3:1 ketogenic diet (3:1 KD) improved food pellet reaching as assessed with the Montoya staircase test (experiment 2).

<p>(A) C5 hemi-contusion injury produced marked impairments in reaching with the ipsilateral forelimb, as illustrated by decreased ability to retrieve pellets in the staircase test. However, at 6 wks post-injury 3:1 KD treated animals retrieved pellets twice as successful with the ipsilateral paw compared to animals fed SD. (B) Animals also displayed a decreased skilled reaching success with the paw contralateral to the lesion. No differences were observed between groups. (C) KD animals were more successful than SD animals in retrieving pellets from the lower wells. Error bars indicate SEM. * p ≤ 0.05 (t-test).</p

Body weight, caloric intake, and blood ketone levels in rats consuming a 3:1 ketogenic diet (KD) or standard diet (SD) after C5 hemi-contusion injury (experiment 1).

<p>(A) During the first week both groups of rats showed reduced food intake but this was more pronounced in the 3:1 KD group on day 1 and 2, which (B) resulted in some additional transient weight loss compared to SD animals. After 2 weeks until the remainder of the experiment, there was no difference in body weight observed between the groups. (C) Blood ketone (ß-hydroxybutyrate) levels increased when consuming 3:1 KD. Error bars indicate SEM. * p ≤ 0.05 (ANOVA repeated measures).</p

KD-induced neuroprotective effect is lost in when MCT transport is inhibited using α-cyano-4-hydroxy-cinnamate (4-CIN).

<p>At day 7-post injury, the total lesion volume (Cavalieri’s Estimator of Morphometric Volume) was decreased in spinal cords of KD fed animals compared to the standard diet group (SD). However, no difference in lesion volume between both groups was observed when animals were treated for 7 days with the MCT inhibitor 4-CIN. Error bars indicate SEM. # p=0.057 (t-test, one tailed).</p

Effect of 3:1 ketogenic diet (KD) on forelimb usage during grooming after C5 hemi-contusion (experiment 1).

<p>A. Scoring system used: Animal’s forepaw 0) was unable to contact any part of the face or head; 1) touched underside of chin and/or mouth area; 2) contacted area between nose and eyes, but not the eyes; 3) contacted eyes and area up to, but not including, the front of ears; an additional 0.5 score was given if the paw reached higher than half of the areas total length; 4) contacted front but not back of ears; 5) contacted area behind ears. (B) 3:1 KD treated rats demonstrated an increased grooming score, compared to SD animals. (C) After 14 wks of 3:1 ketogenic diet treatment (KD), we observed an improved forelimb range of motion compared to standard diet fed animals (SD). 66% of the KD animals reached up to the level of the eye (score 3). Most of the SD animals (88%) could only reach as far up as the nose (score 2). Error bars indicate SEM. * p=0.05 (Chi-square test).</p

Monocarboxylate transporter-1 (MCT1) expression increased in spinal of KD fed rats at 14 days after SCI.

<p>(A) Immunoblot of spinal cord samples probed with antibody to MCT1 showing the expected 42 kDa MCT1 band. As a control for equal protein loading, the corresponding actin signal is shown. (B) The relative MCT1 expression level was significantly higher (1.7-fold) in the KD group compared to the SD group (average of 4 rats). Values are normalized to actin values. Error bars indicate SEM. * p=0.021 (t-test). (C) Triple immunofluorescence confocal image to characterize the localization of MCT1 (green) on glial fibrillary acidic protein (GFAP, red) and glucose transporter-1 (GLUT1; blue) in the spinal cord of a KD fed rat (Spinning disc confocal microscope, 63x, 1.4 NA).</p

Forelimb usage improved under ketogenic diet (3:1 KD) treatment in C5 hemi-contused rats (experiment 1).

<p>(A) The contusion injury produced marked deficits in rearing performance in both groups. Rats fed a 3:1 KD showed increased paw placements of their ipsilateral paw when placed simultaneously with the contralateral paw (both). (B) In a separate cohort of rats (experiment 2) we demonstrated that after switching from to a standard carbohydrate-based diet after 12-weeks of 3:1 KD treatment the forelimb usage remained elevated for 4 weeks compared to SD animals. Error bars indicate SEM. * p ≤ 0.05, ^# p=0.06 (t-test).</p

Ketogenic diet (3:1 KD) improved the grasping and supination component of the pellet reaching movement (experiment 2).

<p>(A-B) The following components were scored: I) Advance; II) Digit extension; III) Pronation; IV) Grasp; V) Supination I; VI) Supination II; VII) Release. On a 3-point scale from 0-1 a score of 1 indicated the presence of the “correct” or “complete” movement, 0.5 the presence of an irregular or incomplete movement and 0 the absence of the movement. (A) Compared to pre-operative scores, (B) C5 hemi-contusion injury dramatically affected reaching components in both SD and KD group. (CD) Grasping and (EF) supination I results of individual animals are blotted. Post-injury KD treatment for 12 weeks resulted in higher average (C) grasping and (E) supination scores based on 10 reach attempts. Dotted line indicates the average score per group. Besides the average score, we also analyzed the highest score achieved (i.e max score) of each animal to assess the animal’s best movement capability during grasping and supination I. (D) None of the SD animals displayed normal grasp movements, however, 50% (6 out of 12) 3:1 KD animals showed a max score of 1 at least once during 10 reach attempts. (F) Despite the severe deficit in supination, almost 3-times as many KD animals (5 out of 12) were capable of normal supination movements compared to SD animals (2 out of 14). Error bars indicate SEM. * p=0.021, ** p=0.003, # p=0.06 (t-test).</p