Experimental Overview
All procedures were approved and performed in accordance with the Institutional Animal Care and Use Committee at The Ohio State University. Aged (22 months) C57BL/6 mice were assessed at baseline and then randomized to a KD (n = 22, 13 female and 9 male) or regular chow diet (n = 15, 7 female and 8 male) ad libitum. Longitudinal assessments were performed at baseline as well as 2, 6 and 10 weeks after the start of the respective dietary intervention. Outcome measures included body mass, hindlimb and all limb grip strength, rotarod latency to fall to assess motor performance, electrically stimulated plantarflexion muscle contractility. Additionally, in the gastrocnemius muscles we used the incremental stimulation motor unit number estimate (MUNE) technique to assess the number of functioning motor units 31, as well as repetitive nerve stimulation at 50 Hz to assess neuromuscular junction transmission 32. At end point (week 10 post intervention), blood samples were collected (submandibular bleed) to assess blood ketone levels (Beta-hydroxybutyrate-BHB). The study design is presented in Fig. 1.
Diet Intervention
The dietary interventions were administered in the morning as needed during the 10-week study. Diets were prepared by Envigo Teklad Diets (Madison, WI) and placed in a small container inside the mouse cages. The High-Fat Ketogenic Teklad Custom Diet (TD.96355) used a fat to protein + carbohydrate ratio. For the regular chow, the 7012 Teklad LM-485 Mouse Sterilizable Diet was used, which is a fixed formula, autoclavable diet, manufactured with high quality ingredients and designed to support the growth of rodents. Typical concentrations of isoflavones (daidzein + genistein aglycone equivalents) range from 300 to 600 mg/kg. 7012 is supplemented with additional vitamins to ensure nutritional adequacy after autoclaving. The ingredients used were: Ground corn, dehulled soybean meal, ground oats, wheat middling, dehydrated alfalfa meal, soybean oil, corn gluten meal, calcium carbonate, dicalcium phosphate, brewers dried yeast, iodized salt, choline chloride, kaolin, magnesium oxide, L-lysine, DL-methionine, ferrous sulfate, menadione sodium bisulfite complex (source of vitamin K activity), vitamin E acetate, thiamin mononitrate, calcium pantothenate, manganous oxide, niacin, copper sulfate, zinc oxide, vitamin A acetate, pyridoxine hydrochloride, riboflavin, vitamin D3 supplement, vitamin B12 supplement, folic acid, biotin, calcium iodate, and cobalt carbonate. Nutrient information and values for the ketogenic diet and regular chow were selected and calculated from ingredient analysis and manufacturer data. The dietary information is presented in the Table 1.
Table 1
Macronutrient composition, energy density and calories from macronutrients.
Macronutrients | Regular Chow (7012 Teklad LM-485) | KD (TD.96355) |
Protein | % | 19.1 | 15.3 |
Carbohydrate | % | 44.3 | 0.5 |
Fat | % | 5.8 | 67.4 |
Fiber | % | 4.6 | 8.8 |
Energy Density | kcal/g | 3.1 | 6.7 |
Calories From: | | | |
Protein | % | 25 | 9.2 |
Carbohydrate | % | 58 | 0.3 |
Fat | % | 17 | 90.5 |
Animal Anesthesia and Preparation
Mice were anesthetized during electrophysiological recordings and during the muscle contractility assessment via inhaled isoflurane delivered at 3–5% for induction and 1–2% for maintenance anesthesia using a SomnoSuite low-flow anesthesia system (Kent Scientific, Torrington, CT). Body temperature was maintained at 37°C with an infrared heating pad (Kent Scientific). To avoid corneal dryness, a petroleum-based eye lubricant (Dechra, Northwich, UK) was applied. Hair of the right hindlimb was shaved (model VPG 6530, Remington, DeForest, WI). The procedures were carried out as we have previously described 31,33,34.
Grip Strength Test
Bilateral hindlimb and all limb grip strength were assessed as previously described using a force transducer (Model GT3, Bioseb SAS BP32025-F-13845 Vitrolles Cedex, Pinellas Park, FL, USA) 35. Mice were grasped and allowed to grip a T-shaped bar connected to the transducer and then were pulled away from the grip meter using a steady and constant motion until grip was lost. For all limb grip, mice were allowed to grip a grid connected to the force transducer and were pulled away from the grip meter using a steady and constant motion until grip was lost. Three trials of hindlimb and all limb grip strength were completed, and the average of the three trials (in grams) was used for analyses.
Rotarod Latency to Fall
Coordination and motor performance were analyzed and conducted using the rotarod test (BX-ROD, Bioseb). To start the test, once the mice were placed on the rod, the rod started rotating at 4 rpm and accelerated at 1 rpm/6 s to a maximum of 40 rpm 36. Three trials were performed at each timepoint and averaged.
Electrophysiological Assessment of Motor Unit Size and Number and Neuromuscular Junction Transmission
Motor Unit Number Estimation (MUNE) was performed to estimate the number of functioning motor units using an approach similar to previous studies in aged mice via a clinical electrodiagnostic system (Cadwell, Kennewick, WA, USA) 31,33. A pair of 28-gauge insulated monopolar needle electrodes (Teca, Oxford Instruments Medical, NY, USA) were inserted subcutaneously into the proximal hindlimb in the region of the sciatic nerve as the cathode and anode for stimulation. A pair of fine wire ring electrodes (Alpine Biomed, Skovlunde, Denmark) were used as the active electrode (placed over proximal gastrocnemius just distal to the knee) and reference electrode (placed over the metatarsal area of the right foot). The ground electrode was placed on the tail (Care Fusion, Middleton, WI, USA). Low frequency and high frequency filters were set at 10 Hz to 10 kHz, respectively. To determine MUNE, first, the peak-to-peak amplitude of the compound muscle action potential was recorded following supramaximal stimulation of the sciatic nerve (constant current: <10mA, duration 0.1 ms). Then, 10 incremental, all-or-none responses obtained during a gradually increasing stimulations were recorded and averaged to calculate the average single motor unit potential (SMUP) amplitude. Then, MUNE was calculated as such: MUNE = Peak-to-peak CMAP Amplitude/ SMUP.
Repetitive Nerve Stimulation (RNS) testing was then performed using the same recording setup as described for MUNE. During RNS, trains of 10 stimulations were delivered at 50 Hz. Amplitude changes between the first and 10th stimulations were calculated using the following formula: % amplitude decrease= [(Amplitude of 10th response − Amplitude 1st response)/Amplitude of 1st response] * 100%.
Plantar Flexion Muscle Contractility
For muscle contractility testing, mice were placed in supine on the testing platform to assess the right hindlimb using an in vivo muscle contractility system (Aurora Scientific Inc, Canada Model 1300A Muscle). The right hindlimb was taped to a rotating foot plate connected to a dual control motor to assess plantar flexion torque. Then, the hindlimb was locked into testing frame connected to the platform base using blunt clamps at the femoral condyles taking care to avoid injury of the fibular nerve at the fibular head. The tibial nerve located in the posterior medial knee was then stimulated using two insulated monopolar electrodes placed subcutaneously (Natus Neurology Inc, Middleton, WI, USA). Stimulation was adjusted (constant current: 0–10 mA, 0.2 ms) to determine the intensity required for a maximal twitch response and adjusted to 120–150% to ensure maximal stimulation. Peak twitch torque was measured after a single 0.2 ms supramaximal pulse-wave stimulation. Tetanic torque was measured using a 200 ms train of stimuli delivered at 125 Hz. This entire process was carried out as previously described 31,33,37.
Blood Collection
Blood beta-hydroxybutyrate and blood glucose were assessed using the Keto mojo B Ketone and Blood Glucose Monitoring System Test Kit. Blood was obtained from the submandibular vein at week 10. The animal did not need to be anesthetized for this process. The mouse was held in one hand using the index finger and thumb which applied the desired pressure to the maxillary vein. The maxillary vein was located along the curvature of the mandible, just below this mark in the groove that runs through the mandible. Using a lancet (needle), firm pressure was applied to the maxillary point, caudal to the eye and ventral to the ear, where the submandibular vein is located, then released until blood flowed. The lancet was kept perpendicular to the bleeding site to avoid injuring the ear canal. Ketone and glucose test strips were then placed under the puncture site using the Keto – Mojo GK + blood glucose & B – ketone dual monitoring system instrument until the desired volume of blood was collected for measurement in mmol/L. Finally, gentle pressure was applied with a gauze over the mandibular area to stop the bleeding of the mouse.
Wet Weight
At the end of the study, the mice were deeply anesthetized before being sacrificed. The gastrocnemius and soleus muscles of the right hindlimb were dissected and removed. Wet weight in grams was recorded on a previously calibrated scale.
Statistical Analyses
Statistical analyzes were performed using the GraphPad Prism 9.5.0 program (GraphPad Software Inc., San Diego, CA, USA). To identify whether there was a trend for an effect of the intervention on body mass, the Eta2 effect size (ƞ2) was used. For the grip test, rotarod performance test, electrophysiology and muscle contractility analyses, two-way ANOVA mixed-effects analysis (time x diet) was used to compare the dietary groups across time points. To determine group differences in blood ketone levels in the intervention group and control group, an unpaired t-test was used. Statistical significance was set at p < 0.05.