Ketogenic Diet as a potential treatment for traumatic brain injury in mice

Traumatic brain injury (TBI) is a brain dysfunction without present treatment. Previous studies have shown that animals fed ketogenic diet (KD) perform better in learning tasks than those fed standard diet (SD) following brain injury. The goal of this study was to examine whether KD is a neuroprotective in TBI mouse model. We utilized a closed head injury model to induce TBI in mice, followed by up to 30 days of KD/SD. Elevated levels of ketone bodies were confirmed in the blood following KD. Cognitive and behavioral performance was assessed post injury and molecular and cellular changes were assessed within the temporal cortex and hippocampus. Y-maze and Novel Object Recognition tasks indicated that mTBI mice maintained on KD displayed better cognitive abilities than mTBI mice maintained on SD. Mice maintained on SD post-injury demonstrated SIRT1 reduction when compared with uninjured and KD groups. In addition, KD management attenuated mTBI-induced astrocyte reactivity in the dentate gyrus and decreased degeneration of neurons in the dentate gyrus and in the cortex. These results support accumulating evidence that KD may be an effective approach to increase the brain’s resistance to damage and suggest a potential new therapeutic strategy for treating TBI.


Scientific Reports
| (2021) 11:23559 | https://doi.org/10.1038/s41598-021-02849-0 www.nature.com/scientificreports/ studies suggest that KD induces anti-inflammatory effects 16 . Studies have reported that KD, and in particular, the ketone metabolite beta-hydroxybutyrate suppresses activation of the NLRP3 inflammation in response to several structurally unrelated NLRP3 activators 16 and improves the brain's ischemic tolerance 17 . KD management has also been shown to reduce activated microglial expression 18 . While mechanism of KD in neuroprotection is unknown, past reports found that KD increases glutathione level 19 and Uncoupling protein (UCP) 20 in cells after brain injuries, decreasing Reactive Oxygen Species (ROS). A possible mechanism by which KD could be inducing neuroprotective effects is through the alteration of SIRT1 expression. SIRT1 has a role in developing the hippocampus by activating Akt and inhibiting GSK3 and is involved in various physiological processes such as oxidative stress response, genetic silencing, genome stability, and cell life extension 21,22 . The Sirtuin family of proteins is also active in the hypothalamus, where it plays a role in regulating circadian rhythm, endocrine pathways, and appetite [23][24][25][26] . Recently, several studies have shown that SIRT1 plays a significant role in induced neuroprotection following caloric restriction (CR) 27,28 . CR has also been linked to reduced tau phosphorylation and maintenance of hippocampal neurons 29 , and related Tau pathology is known to be implicated in different types of TBI 30,31 . While CR has been shown to increase ketone bodies in the blood, KD does this at a higher level 32 . This is important to note as high fat diet and increased circulating ketone bodies have been shown to activate SIRT1 33 . Thus, the effects of SIRT1 following KD, as in our model, may be more pronounced than in previous models utilizing CR. Our present study utilizing adult male ICR mice coincides with past rodent studies addressing SIRT1 34-36 . In the present study, we utilize a closed head weight drop model of murine mTBI to test the cognitive, cellular, and molecular effects of up to 30 days of KD management following injury. We report that KD initiated after mTBI ameliorated the cognitive deficits in spatial and visual memory, as well as cellular changes in neurons and glial cells induced by the injury. Our model also shows that KD sustained the levels of SIRT1 expression which were decreased with injury.

KD increased the level of ketone bodies in blood.
One-way repeated ANOVA revealed a significant main effect of time [F (3,63) = 28.31, p = 0.000, η 2 = 0.57] and group [F (3,21) = 49.24, p = 0.000, η 2 = 0.88] as well a time by group interaction [F (9,63) = 9.81, p = 0.000, η 2 = 0.58]. Consecutive simple effects analysis with Sidak test indicated that the level of ketone bodies in the blood of KD mice was significantly higher than the level in mTBI and control mice at the 3-day time-point (both p = 0.000), 7-day time-point (both p = 0.000), and 30-day timepoint (both p = 0.001). Similarly, the blood ketone levels in the mTBI + KD group were persistently higher than that of the mTBI and control groups at the 3-day time-point (both p = 0.000), 7-day time-point (both p = 0.001), and 30-day time-point (p = 0.022 and p = 0.018, respectively). See Fig. 1  SIRT1 expression within the cortex-one-way ANOVA revealed significant between-group differences in SIRT1 expression [F (3,21) = 6.26, p = 0.003, η 2 = 0.47]. Gabriel's post-hoc analysis showed that the mTBI group had significantly lower SIRT1 levels than the mTBI + KD group (p = 0.002). In addition, the mTBI group showed a marginally significant trend toward lower SIRT1 levels than the than the control (p = 0.090) and KD (p = 0.077) groups. See Fig. 3B.
Ketogenic Diet prevents mTBI-induced neuronal loss. The number of NeuN + neurons in the cortex and dentate gyrus-one-way ANOVA revealed significant between-group differences in the number of NeuN + neurons both within the cortex [F (3, 16) = 5.06, p = 0.012, η 2 = 0.49] and the dentate gyrus [F (3, 16) = 8.51, p = 0.001, η 2 = 0.61]. Gabriel's post-hoc analysis demonstrated that the total number of NeuN + neurons within the cortex was significantly lower in the mTBI group than in the control (p = 0.018) and mTBI + KD groups (p = 0.028). The total number of NeuN + neurons within the dentate gyrus was significantly lower in the mTBI group than in the control (p = 0.008), KD (p = 0.012), and mTBI + KD (p = 0.002) groups. See Fig. 4(A-C).
GFAP Morphology in the cortex and dentate gyrus-one-way ANOVA revealed significant between-group differences in GFAP Morphology in the dentate gyrus DGH-[F (3,16)

Discussion
TBI is a leading cause of death and long-term disability in the developed world, with more than 10 million people suffering worldwide every year 37 . The majority of these TBIs (80-95%) are mild in nature 7,8 . TBI symptoms can occasionally resolve within the first year after injury, but up to 70-90% of patients continue to manifest prolonged and often permanent neurocognitive dysfunction. In light of the growing reports suggesting beneficial effects of KD in many neurological disorders [13][14][15] , the primary goal of this study was to assess the benefits of KD in mTBI.
Our results suggest that KD may be a vital treatment modality, mitigating TBI-induced cognitive impairments, neuronal loss, and neuroinflammation in the closed-head mTBI mouse model. We delivered a mild TBI to mice followed by up to 30 days of KD or SD. We were able to confirm that mice who received KD (with or without mTBI) demonstrated a prominent increase in ketone bodies compared to mice fed SD at 3, 7, and 30 days after the diet. Previous studies have demonstrated that mice challenged with mTBI show cognitive impairments in visual and spatial memory 10,38 . Our previous published studies as well as the present study, ruled out the possible involvement of anxiety (as assessed by EPM) in the cognitive performance of the injured mice. This study's results replicated the previous findings regarding both visual and spatial memory while demonstrating that KD significantly ameliorated these cognitive deficits. These results support previous studies on the benefits of KD on brain injury in the short-term (7 days); rats who were fed KD exhibited evidence of neuroprotection after head trauma in the cortex and hippocampus 39 and improvement in motor performance   18 , KD administration has also been shown to reduce brain edema and cellular apoptosis 72 h after TBI 40 and has demonstrated anti-tumor effects within the brain in rodent models 41 .
To better understand the molecular effects of TBI and KD, we evaluated SIRT1 levels in two brain regions, cortex and hippocampus, which play crucial roles in memory formation. TBI reduced the levels of SIRT1 in cortex and in the hippocampus 30 days post-injury which was ameliorated by KD, suggesting a potential mechanism contributing to cognitive impairment and improvement in cognitive symptoms when given KD 42 . In contrast, at 7 days post-injury SIRT1 levels were not reduced in the cortex and hippocampus. This supports past findings showing an elevation in SIRT1 levels 1 day prior to ischemia that gradually decreased over a period of 7 days 36 . Our study's results align with prior studies of intermittent fasting and caloric restriction for 30 days post-injury 28 and with research involving KD and SIRT1 in health-enhancing, aging, longevity, and neurodegeneration in animal models 43,44 .
We have previously reported that our mTBI model decreases the neuronal survival 3 weeks post injury 45 . Similarly, in the present study, we have shown a significant increase in neuronal cell death that persists 30 days following injury in both cortex and hippocampus. KD prevented this neuronal cell death. These results are consistent with an in vitro study that found the ketone body beta-hydroxybutyrate reduced axonal degeneration in diffuse axonal injury 46 . Additionally, we saw a marked increase in reactive astrocytes in hippocampus 30 days following injury, which was ameliorated by KD. We have previously reported that our mTBI model induces fundamental neuroinflammatory changes, including elevations in astrocyte reactivity, pro-inflammatory cytokine TNF-α levels, and expression of genes involved in inflammatory processes in several regions of the brain 44,47-50 . We can conclude that KD prevented the injury-induced neuro-inflammation, which is in agreement with previous studies that reported KD inhibited NLRP3 inflammasome activation, thus exerting neuroprotective effects 17 .
Our closed head injury mouse model of mild TBI was able to recapitulate the cognitive deficits observed in human TBI patients 51,52 and showed that ketogenic diet beginning following injury could protect against injury-induced memory loss. At the cellular level, we have demonstrated that mTBI induced neuronal cell death, astrocyte and microglial activation (neuro-inflammation), which were prevented in injured mice treated with KD. Our analysis of changes in SIRT1 suggests a viable molecular mechanism by which KD may be improving cognitive outcomes. Our results support accumulating evidence that KD may represent an important nonpharmaceutical treatment against the long-term molecular and cellular impacts of mTBI, which may ultimately improve cognitive symptoms and quality of life for TBI patients.

Methods
Experimental procedures. Mice were subjected to mTBI, and a ketogenic diet (KD) was initiated immediately for the following three timelines: 3 days, 7 days, and 30 days. Ketone bodies in the blood were measured 0, 3, 7, and 30 days post-injury. Behavioral tests were performed at 7 or 30 days following mTBI in separate groups of animals. Western blot analysis was carried out on brain tissue collected from 7 and 30 days post mTBI. Immunohistochemical staining assessments were performed on brains that were collected 30 days post-injury. The timeline of the experimental procedures following exposure to mTBI is shown in Fig. 6.

Animals. The Sackler Commission on Animal Experimentation ethical committee approved the study and
animal protocol 01-19-058 according to the Guidelines for Animal Experimentation of the National Institutes of Health (DHEW publication 23-85, revised, 1995). The study complies with ARRIVE guidelines.
Male ICR mice, aged 6-7 weeks, 30-40 g body weight, were acquired from Envigo RMS Israel. Mice were housed at 4-5 per home cage under a constant 12 h light/dark cycle, at room temperature (22 ± 2 °C) in a standard Figure 6. Study timeline. Animals were exposed to mTBI or sham and checked for ketone bodies in the blood at days 0, 3, 7 and 30. Mice were then fed KD/SD for 3, 7, or 30 days. Behavioral tests to assess behavior and cognitive abilities were carried out at 7 and 30 days following mTBI in separate cohorts. Western blot analysis to assess changes in SIRT1 levels following mTBI and diets management was performed at 7 and 30 days post-mTBI. Immunohistochemical staining to evaluate neurodegeneration and neuroinflammation was performed at 30 days following the mTBI challenge. . Water and food was provided ad libitum, and the cages bedding, sterile sawdust, was replaced once per week. All mice were acclimatized to the facility for three days following transport and then moved into the experimental testing room for three days prior to experimentation. Animals were utilized only once throughout the study in either behavioral, biochemistry, or immunocytochemistry tests. Each animal in a given group was tested only once in order to avoid the possible confounding effect of behavioral testing. The number of animals evaluated in each assessment group and the measurement times performed was based on analysis of variance in our previous studies.
Mouse closed-head mild traumatic brain injury. Mild Traumatic Brain Injury (mTBI) was induced according to the closed-head weight-drop model, as employed in our previous studies 53,54 . The device consisted of an aluminum tube (80 cm in length and 13 mm in diameter). In the pre-injury stage, mice were anesthetized by inhalation of isoflurane and placed under the device on a sponge, the sponge supported the head of the mouse allowing some anterior-posterior motion, but no rotational head movement at the moment of impact. A metal weight (50 g) was dropped from the top of the tube to strike the head on the right temporal side between the ear and corner of the eye. Sham mice received anesthesia and were placed on the sponge for an equivalent length of time, but no weight was dropped. This model was chosen because it simulates traumatic head injuries such as road accidents or falls, as it imposes a diffuse and non-specific injury. Our model, while subject to a certain amount of variability due to the natural anatomic variation, still maintains a high degree of TBI injury similarity among all mice as seen in previous neuronal loss models 53 57 . The diets and water were provided ad libitum for 3, 7, and 30 days after the injury. Both diets were stored at 4 °C, KD, which had a solid, butter-like texture, was changed every day, and SD pellets were changed twice per week.

Measurement of Ketone Bodies. Ketone bodies in the blood were measured using the Precision Xtra
Blood Glucose and Ketone Monitoring System (Abbott, Columbus, OH) 60 . Mice were anesthetized with isoflurane and tails were cut approximately 0.2 mm from the end, a drop of blood was squeezed directly into a testing strip attached to the measuring instrument. Group sizes were as follows: control (n = 8), mTBI (n = 6), KD (n = 6), and mTBI + KD (n = 5).
Elevated plus maze. The elevated plus maze (EPM) was used to evaluate anxiety-like behavior 61  Novel object recognition. The novel object recognition (NOR) task assesses recognition and visual memory 63 . This paradigm relies on rodents' natural tendency to investigate novel objects within their environment rather than known ones. NOR evaluates whether a mouse is able to discriminate between a familiar and a novel object. The testing arena is a square surface (60 cm × 60 cm) with high walls (20 cm). The test consists of three 5 min sessions, separated by 24 h. On the first day, mice were individually put in the empty arena for habituation for 5 min. On the second day, the mice were exposed to 2 identical objects within the arena for 5 min. On the third (experimental) day, one of the familiar objects was replaced with a novel object, and mice were allowed to explore the arena again for 5 min, during which time spent near familiar and novel object was measured. The arena was cleaned with 70% ethanol between subjects. An Aggelton index was calculated as follows: (time near new novel object − time near familiar object)/(time near new novel object + time near familiar object) 64 . A higher Aggelton index indicates advancement in recognition memory. Animals near the objects less than 10% of the total test time (i.e., less than 30 s next to the two objects together) were excluded from statistical calculations, as exploration under this amount of time does not allow estimation of subjects' visual memory. Group sizes were as follows: control (n = 19), mTBI (n = 18), KD (n = 23), and mTBI + KD (n = 24). www.nature.com/scientificreports/ Y-maze. The Y-maze paradigm was used to evaluate spontaneous exploration, responsiveness to novel environments, and spatial memory function, as previously described 65 . This test relies on the preference of rodents to explore new environments rather than familiar ones. The Y-maze consists of a three-armed black plexiglass maze with arms separated by 120°. Each arm was identical (8 × 30 × 15 cm); however, different spatial cues were placed in each arm (i.e., a triangle, a square, or a circle). In the first session of the test, the mouse was put in the arena's start arm (chosen randomly) and allowed to explore another arm while the third arm was blocked for 5 min. The mouse was then returned to its home cage for 2 min. In Immunohistochemistry. Immunohistochemistry studies were performed hippocampal (dentate gyrus) and temporal cortex tissue sections obtained from animals euthanized on day 30 post-injury. Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and underwent transcardial perfusion with 10 ml phosphate-buffered saline (PBS) followed by 20 ml of 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. Brains were removed, fixed overnight in 4% PFA, and then placed in 1% PFA. Neuroscience Associates (Knoxville, TN) oriented the brains into a multiblock, collected 35 μm sections sequentially through the brains, and performed the floating section staining and mounting (antibodies detailed in Table 2). Microscopy was performed with a Fluoview 3000 laser scanning confocal microscope (Olympus, Waltham, MA). Target locations were determined on a stitched map with only Hoechst 33342 staining captured. For all analyses, regions of interest were selected on the map depicting only the nuclear staining and blinded with respect to groupings. These regions were then collected by multi area time lapse in sequence with the Fluoview 3000 software without intervention. Images, centered on coronal sections at approximately − 2.9 mm from Bregma, were collected as Z stacks and maximum Z projections with constant illumination (405, 488, 561, and 640 nm diode lasers) and detection parameters. Automated analysis for morphology, intensities, and numbers of cells was conducted using cellSens (Olympus, Waltham, MA, USA) and ImageJ 66,67 . Changes in the architecture of astrocytes were determined by batch processing of all images collected with the aid of ImageJ. All of the following steps were iteratively executed for each micrograph in a single macro. Each image was analyzed for the channel containing GFAP staining. Background was subtracted with ImageJ's built-in rolling ball process, automated thresholding was accomplished with the built in RenyiEntropy algorithm, and noise was removed using the ImageJ despeckle routine. The image was then skeletonized with the ImageJ plugin andfeatures that were too small to be relevant were removed by the particle remover plugin. The analyze skeleton plugin generated the process lengths per cell. These routines have previously been shown to identify reactive morphology 66,67 .
Frames included in figures are correct concerning orientation, i.e., dorsal at the top, left, and right. Confocal scanning was rotated 30° to optimize the framing of regions of interest. Group sizes were as follows: control (n = 5), mTBI (n = 5), KD (n = 5), and mTBI + KD (n = 5).
Western blotting. To assess the cortical and hippocampal SIRT1 levels, brains were dissected following cervical dislocation at 7 and 30 days post-injury. The cortex and hippocampus were separated and frozen in liquid nitrogen, then stored in − 80 °C. Prior to analysis, brains were homogenized in lysis buffer (Tissue Protein Extraction Reagent, Pierce, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Halt Protease Inhibitor Cocktail, Sigma Aldrich, St. Louis, MO, USA) using a Teflon pestle homogenizer. Homogenates were centrifuged for 15 min at 4 °C 14,000 r/min, supernatant liquids were separated from the precipitates and stored at − 80 °C. Sample buffer was added to the samples and then stored at − 18 °C. Prior to analysis, samples were heated to 90 °C for 3 min and 30 µl of each sample was then loaded and run on 4-20% Mini-Protean TGX gels (Bio-Rad, Hercules, CA, USA) followed by transfer onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) by a transfer system (Trans-Blot Turbo, Bio-Rad, Hercules, CA, USA). Afterward, blots were blocked for 1 h at room temperature, with Tris-buffered saline, containing 0.01% Tween-20 and 5% BSA or powdered milk. Membranes were then incubated overnight at 4 °C with a mouse primary anti-SIRT1 antibody (Abcam, Cambridge, UK, ab10304, 1:500) and washed with TBS. Membranes were then incubated at room temperature for www.nature.com/scientificreports/ 1 h with goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, 115-035-003, 1:10,000). Bands were then exposed using enhanced chemiluminescence with ECL (Millipore, Billerica, MA, United States) for 1 min by Viber Fusion FX7 imaging system (Viber Lourmat, France). Densitometry analysis of the detected signal was performed using ImageJ software. Uniform loading was verified by stripping and reprobing with a mouse primary α-tubulin antibody for 30 min at room temperature (Santa Cruz Biotechnology, Dallas, TX, sc-53030, 1:10,000), then conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, 115-035-003, 1:10,000). The ratio of SIRT1 and α-tubulin (TUB) determined the value of each sample. Averages of control values in each membrane were set to 1, and all other samples were calculated accordingly 28 . Figure 3A,B shows cropped blots, the membrane was cropped immediately after the transfer stage due to the usage of two different antibodies on the same blot membrane, SIRT1 band size was at 110 kDa and α-tubulin band size was at 55 kDa, enabling the process of cropping and incubating two different antibodies due to the distantness from each other without risk of damaging the membranes pristine state. Fulllength blots/gels are presented in Supplementary Figs. 1-4. Group sizes were as follows: control (n = 7), mTBI (n = 6), KD (n = 5), and mTBI + KD (n = 7).

Statistical analysis.
Statistical analysis was carried out using IBM SPSS version 24.0. All values are presented as the mean ± standard error of the mean (SEM). Statistical analysis included data imputation in order to maximize power, followed by one-way ANOVA, repeated measures ANOVA, or two-way ANOVA where appropriate. Gabriel and Sidak (α = 0.05) tests were used as post hoc tests. Partial eta squared (η 2 ) was calculated to show effect size. Significance was determined as a two-sided p < 0.05. Descriptive statistics for blood ketones are provided in supplementary files,

Data availability
All data supporting this study and its findings are available within the article or from the corresponding author upon reasonable request.