G93A transgenic mice and dietary conditions
Male SOD1-G93A mutant transgenic mice (stock #002297) were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in our transgenic mouse facility to generate SOD1-G93A mice and wild-type (WT) control littermates. Of the Twenty-seven SOD1-G93A transgenic ALS mice identified for this study, 11 males were selected. Only males were used because of the background and gender effects on survival in this mouse model of ALS . At 50 days of age animals placed on either a ketogenic diet (caloric composition, fat 60%, carbohydrate 20%, protein 20%) or a standard rodent laboratory diet (fat 10%, carbohydrate 70%, protein 20%). Both diets contained equal percentages of cholesterol per gram (Research Diet, Inc. New Brunswick, NJ). Mice were housed on a 12 hour light, 12 hour dark cycle and allowed ad libitum access to food. Mice were weighed at the start of treatment, day 89 (pre-symptomatic) and at study endpoint. The study endpoint was defined as meeting any one of the following conditions: no spontaneous breathing or movement for 60 seconds with no response to pain; the animal is unable to right itself after 10 seconds following a push over; or complete hind limb paralysis.
Mice were sacrificed by cervical dislocation. Spinal cords were dissected and snap frozen and stored at -80°C. Blood was collected, and serum was obtained after 15 minutes of clotting and centrifugation at 4°C. The Institutional Animal Care Committee of Mount Sinai School of Medicine reviewed and approved all experimental protocols used in this study.
Motor Function Assessment
SOD1-G93A mice were tested on the accelerating rotarod (7650 Ugo Basile Biol. Res. App., Comerio, Italy) as described previously . Mice were given 5 days of practice to become acquainted with the rotarod and human handling. The examiner was blinded as to treatment group. At 85 days of life and continuing until death, the time the mouse could stay on the rotating rod was measured. Only animals that could successfully maintain balance for 180 sec for 2 successive days were included to ensure homogeneity of motor performance in all animals at onset. (n = 10: 5 KD; 5 standard feed). Testing was conducted during the last 4 hours of the day portion of the light cycle in an environment with minimal stimuli (noise, movement, changes in light or temperature).
Histology and stereological analysis
For stereological analysis, 10 serial coronal sections (12 μm thick) were cut 350 μm apart through the lumbar (L3 to L5) spinal cord of each animal (WT controls; n = 5) and SOD1-G93A mice fed either the KD (n = 6) or standard diet (n = 4). The sections were mounted onto positively charged glass slides (Superfrost Plus, Fisher Scientific) and Nissel stained. Large (>25 μm), Nissl-stained neurons were counted within the ventral horns under a light microscope at a magnification of × 200. These counts were within a homogenous structure, making the tenets of stereology valid. Nissel-stained neurons were counted using the Neurolucida system at a magnification of × 250 in both ventral horn areas from six L3-L5 tissue sections of the spinal cord of each mouse, with size discrimination into diameter classes of >25 μm. All cells were counted from within the ventral horn below a lateral line across the spinal cord from the central canal. Correction for tissue section thickness was made in all specimens.
Serum ketone measurement
There are three ketone bodies: D-β-3-hydroxybutyrate (DBH), acetone, and acetoacetate. The concentration of all three bodies represents the total concentration of ketone bodies in the blood. The concentration of ketone bodies in SOD1-G93A mice was determined using the Autokit 3-HB and Autokit Total ketone bodies respectively (Wako, Osaka, Japan), according to the manufacturer's instructions. Serum (3 μl diluted 5- or 10-fold) was incubated with 202.5 μl of reagent R1 in a 96 well plate, for 5 minutes at 37°C. Then 67.5 μl of reagent R2, was added for another 5 minutes and incubated at 37°C. The rate of Thio-NADH production was measured spectrophotometrically at 405 nm using a Dynatech MR5000 spectrophotometric 96 well plate reader (Coulton). A standard curve was established using two-fold dilutions of the Total Ketone Body calibrator (Wako) starting at 300 μmol/L. Samples were diluted 0, 3, 5 or 10 fold to fit within the standard curve which was linear over six 2-fold dilutions.
Mixed spinal cord cultures were prepared according to Spalloni et al. [16, 23]. Briefly, spinal cord cultures were prepared from E14 embryos dissected from a pregnant WT female mated with a SOD1-G93A male. Each spinal tube was dissected, removed from the meninges, and incubated for 10 min in 0.25% trypsin/EDTA at 37°C and then dissociated by gentle trituration with a fire-polished pasteur pipette. The resulting mixed neurons were plated on poly-D-lysine-coated 8 well chamber slides (Nalge Nunc Inc., NY) in D-MEM/F12 supplemented with 10% fetal bovine serum (FBS) and maintained at 37°C in a humidified atmosphere of 5% CO2. Two to three hours after plating, the medium was replaced with neurobasal medium supplemented with 2% B-27, 0.5 mM glutamine, and 1% penicillin/streptomycin.
Rotenone, malonic acid, sodium azide and DHB were obtained from Sigma-Aldrich, St Louis, MO. Ten-day-old neuron cultures were exposed to varying concentrations of rotenone or malonate with or without 5 mM DHB for 48 hours. Cell viability was measured by ATP production level using CellTiter-Glo® luminescent cell viability assay kit according the manufacturer's instructions (Promega Corp Madison, WI).
Cytotoxicity was determined by the amount of lactate dehydrogenase (LDH) activity released in the cell culture media using CytoTox96® Non-Radioactive Cytotoxicity Assay according the manufacturer's instructions (Promega Corp).
Mitochondria preparations and ATP measurements
Mitochondria isolation and ATP measurements were performed using the mitochondria isolation kit and an Adenosine 5'-triphosphate (ATP) Bioluminescent Assay kits respectively according to the manufacturer's instructions (Sigma). Spinal cords from WT and Tg SOD1-G93A animals, 8 weeks of age, were dissected on ice and the tissue was quickly grinded in buffer A with a 3 ml Teflon pestle with 10 -15 strokes. After several centrifugation steps aimed at enriching mitochondria fraction, the final mitochondria pellet was resuspended in isolation buffer A (10 mM HEPES, pH7.5, 200 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 1 mg/ml Albumin). Protein content was determined using the BioRad protein determination kit. For ATP generation measurements, assays were prepared in 100 μl total volume in a Fluorotrac 200 96 well plate (Nunc). Each assay contained buffer A (35 mM K-PO4, pH7.5, 10 mM Mg Acetate, 180 mM sucrose, 1 mM EDTA, 0.1% BSA, 50 mM pyruvate) with freshly added 100 μM ADP, 100 μM P1, P5-Di(adenosine) pentaphosphate, 150 μg/ml luciferin and 1.2 μg/ml luciferase, in the presence or absence of 5 mM DHB and the various inhibitors. The assays were initiated by adding a 10 μl volume of the mitochondrial preparation containing 30 μg of total isolated mitochondria. The luciferase light emission was recorded every minute for 10 min by Fusion™ Universal Microplate Analyzer (Perkin Elmer, MA).
The 10-day-old neuron cultures were treated with drug for 24 hours and cultured with fresh drug free culture media for another 48 hours with or without 5 mM DHB. The cells were fixed with 4% paraformaldehyde and processed to immunocytochemistry with anti-NSE and anti-SMI32 double staining. Total neurons were identified as NSE positive neurons, and motor neurons were regarded as SMI32 positive neurons. The SMI32 positive neurons were counted microscopically at a magnification of × 250 in as least 5 randomly selected fields. The examiner was blind to the treatment.
Statistical analyses were performed using SigmaStat (version 3.0, SPSS Inc., Chicago, IL). Independent measures t-tests were used to compare endpoint serum contents of DHB and total ketone bodies in control vs. ketogenic diet group. Repeated measures t-test was used to compare ATP synthesis rate data obtained from neuron cultures incubated with and without addition of DHB. Deterioration of motor function (rotarod testing) was assessed by the Kaplan-Meier survival analysis (Mantel-Cox log rank test) with "failure" defined as inability of an animal to achieve at least 50% of its baseline motor performance. This endpoint was chosen because it is a measure that is distinctly related to entry criteria, is the midline of an inexorable decline in strength, and is not influenced by observer bias and animal adaptive skills used when the animal is very weak (e.g. holding on to the rotating rod without walking). A nonparametric time to failure analysis was chosen since a two-way repeated measures ANOVA could not be performed because the data failed both normality and equal variance tests. Effect of ketogenic diet on longevity was also assessed by the Kaplan-Meier survival analysis (Mantel-Cox log rank test) with "failure" defined as death of an animal. Animals' body weight data were analyzed by a two-way repeated measures ANOVA with Huynh-Feldt correction. Motor neuron counts data and SMI32 positive neuron counts data from immunochemical evaluation were analyzed by one-way ANOVAs. Data on ATP and LHD levels in the presence of rotenone and malonate were analyzed by two-way ANOVAs. ANOVA tests were followed, when significant, by the Student-Newman-Keuls multiple comparison tests. Student-Newman-Keuls test was chosen for post hoc multiple comparisons due to its generally higher sensitivity compared to the Bonferroni test. In all tests, results with probability values less than 0.05 were considered statistically significant. Presented data are shown as mean ± se, unless otherwise noted.