Chemicals were purchased from Sigma/Merck (Darmstadt, Germany) unless stated otherwise.
Female CD-1 mice (29–32 g, Charles River) were used for the experiments. They were kept in standard cages, under 60% humidity, 22 °C temperature, and a 12 h-light/dark cycle. Food and water were available ad lib. The study was registered with the local animal committee (Regierungspräsidium Darmstadt). In accordance with GV-Solas guidelines, all procedures were designed to minimize the suffering of the experimental animals.
Mice were randomized to study groups using a computer program for random number generation. In total, 173 mice were used for this study. 24 experiments could not be followed through because of surgical problems (insufficient blockade of the MCAO, continuous bleeding during reperfusion), and the mice had to be sacrificed. In 17 experiments, analytical problems caused a failure to obtain data (lack of perfusion in the microdialysis probe, problems during sample work-up and GC–MS measurements). Thus, the results shown in Figs. 1, 2, 3, 4, 5 and 6 were obtained from 132 successful experiments (with an average of eight experiments per group). Separate experiments were performed for the generation of figures, except results in Figs. 6 and 7 that were from the same group of animals.
For surgery, animals were anesthetized with isoflurane (induction dose 5%, maintenance dose 2% v/v) in synthetic air (Air Liquide, Düsseldorf, Germany). Self-constructed, Y-shaped, concentric dialysis probes with a molecular weight cut-off of 10 kDa were stereotaxically implanted into the hypothalamus with the following coordinates (from bregma): AP − 1.5 mm, L + 0.5 mm, DV − 3.8 mm according to . Glass ionomer eluting cement (PermaCem Smartmix Dual, Dental Milestone, Hamburg, Germany) was used to fix the probe on the skull (for further details, see ). Probes were implanted at least 18 h before each experiment to allow recovery to stabilize . Microdialysis was performed on the next day with a perfusion fluid (aCSF) containing 147 mM NaCl, 4 mM KCl, 1.2 mM CaCl2 and 1.2 mM MgCl2. The perfusion rate of the microinjection pump was 2 µL/min. The collection intervals were 15 min. Data are given as absolute levels not adjusted for probe recovery.
Glucose and lactate concentrations in microdialysates were determined by a colorimetric method (530 nm) using an ISCUSflex Microdialysis Analyzer (M dialysis AB, Solna, Sweden).
Transient Middle Cerebral Artery Occlusion (t-MCAO)
The procedure was performed as previously described [20, 21]. Briefly, mice were anesthetized using isoflurane (2% in synthetic air), their body temperature was kept constant using a thermostatic device, and buprenorphine (0.1 mg/kg i.p.) was injected 15 min before performing surgery (this injection was repeated 8 h later). After a paratracheal incision, a silicon suture (Doccol®, Redlands, California; size 6–0) was inserted into the A. carotis communis and advanced into the middle cerebral artery (MCA), where the silicon top of the filament blocked perfusion of the vessel. Cerebral blood flow was monitored with a laser-Doppler monitoring device (Moor Instruments, Devon, UK) to ascertain ischemia (< 15% of blood flow vs. basal). After 90 min, the suture was removed to allow reperfusion. Mice were sacrificed under isoflurane anesthesia either after 60 min or after 1, 3 or 7 days.
Neurological deficits were determined by behavioral testing in the morning before surgery and 24 h after MCAO. The “Chimney test” (modified from ) was performed for each mouse three times before and after surgery. A mouse was placed head first at the entry of a tube (200 mm long and 40 mm diameter). When the mouse reached the bottom of the tube, the tube was raised to an angle of 45 degrees. All mice reacted by walking backwards. The time needed to climb out of the tube was measured for a maximum of 120 s. The “Corner test” was used as described . Mice were placed in a corner (30° angle) and the chosen sides to leave the corner were counted. Each mouse was tested for one trial (maximum time 120 s) before and after surgery. The laterality index (LI) was calculated: (left turns–right turns)/total number of turns . After 24 h, we also calculated a neurological score from animal behavior. Details of the scoring procedure are given in the Suppl. Table 1.
High-Resolution Respirometry in Isolated Mitochondria
After decapitation, the brain was immediately dissected from the skull, the cerebellum was removed and the brain divided into hemispheres. From each hemisphere the frontal part of the brain (≈100 mg) was separated and homogenized in 2 mL MiR05. In addition, a protease inhibitor cocktail (PI) was added to the medium (cOmplete Tablets EASY pack, Roche, Mannheim, Germany). The homogenate was centrifuged twice to remove all cell debris (1400×g, 7 min, 4 °C). The purified supernatant was then centrifuged again (10,000×g, 5 min, 4 °C), the resulting pellet containing the mitochondria was resuspended in 1000 µL MiR05 + PI and centrifuged once again (1400× g, 3 min, 4 °C). Finally, the supernatant was centrifuged one more time (10,000×g, 5 min, 4 °C) and the pellet resuspended in 250 µL MiR05 + PI.
Mitochondria from ischemic and contralateral hemispheres were put into parallel chambers of the respirometer. Each chamber was filled with 2.4 mL MiR05 medium according to manufacturer’s instructions and kept at 37 °C with constant stirring (750 rpm). After 30 min equilibration and subsequent air calibration, 80 µL of the mitochondrial suspensions were injected into the closed chamber. The remaining mitochondria were frozen in liquid nitrogen for protein determination with the Bradford assay. After equilibration, a solution containing pyruvate (5 mM) and malate (1 mM), two substrates linked to complex I (CI), was injected into the chamber (LEAK-state, non-phosphorylating resting state). Then, ADP (2 mM) was added to stimulate oxidative phosphorylation (OXPHOS; ADP-stimulated and CI-linked respiration). To induce the full ADP-stimulated respiration, succinate (10 mM), a CII-linked substrate, was injected (OXPHOS capacity). To verify the integrity of the outer mitochondrial membrane, cytochrome c (10 µM) was added; mitochondria whose respiration increased by more than 15% upon cytochrome c addition were discarded. The maximum capacity of the electron transfer system (ETS) was determined by the stepwise titration of the uncoupler FCCP (state E). To see the isolated CII respiration, the complex I inhibitor rotenone (2.5 µM) was added (CII-linked substrate state, uncoupled). After inhibition of complex III by antimycin A (2.5 µM), the residual oxygen consumption (ROX) remains, which is used to correct the mitochondrial respiration states. Ascorbate (2 mM) and tetramethyl-phenylendiamine (TMPD, 0.5 mM) are artificial electron donors that induce maximum cytochrome c- oxidase (complex IV, CIV) respiration by reducing cytochrome c. Ascorbate regenerates TMPD and is injected first. At the end of the experimental run CIV is inhibited by a high concentration of sodium azide (120 mM). The chemical background as well as ROX remains. To obtain the CIV activity this value has to be subtracted from the total measured oxygen flux (for further details, see ).
Blood plasma and brain samples were harvested immediately after decapitation of mice, frozen in liquid nitrogen and stored at− 80 °C until metabolites were measured by GC–MS. Brain homogenates were extracted using Folch’s procedure, the aqueous supernatant was dried under a stream of nitrogen, and the dry residues were derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS) (99:1). In plasma samples, proteins were precipitated by addition of methanol/water (9:1), centrifuged, and the supernatants were treated as described above.
Samples were measured on an HP-6890 Series GC-System (Hewlett Packard®, Palo Alto, California) coupled to an Agilent Mass Selective Detector 5973 (Agilent®, Waldbrunn, Germany) and an Agilent® Autosampler 7683. We used a VF-5MS capillary column (30 m × 0.25 mm inner diameter) (Varian Technologies®, Palo Alto, CA) with a silylated precolumn (5 m). After the qualitative analysis of the metabolites (spectra adjusted to N.I.S.T. database), we established single ion monitoring (SIM) parameters and used them for quantification of glucose, BHB, citrate, succinate, fumarate and malate. The calculations were done with internal and external standard methods.
If not indicated otherwise, data are presented as means ± SEM of N (number of animals). All data were tested for normal distribution by the Kolmogorov–Smirnov test (GraphPad Prism 5.03). Potential outliers (> 2 SD) were identified by the Grubbs test (https://www.graphpad.com/quickcalcs/grubbs). Sample size was calculated by the formula N = 2 SD2 × power index/delta2. Based on many years of experience, an SD of 20% was expected for metabolite measurements and a treatment effect of 25% was defined as goal of the study. The value for the power index (α = 0.05, two-sided; ß = 0.2; 80%) was taken from the book „Intuitive Biostatistics “ by Harvey Motulsky (Oxford University Press, 1995). Treatment effects on activity changes of mitochondrial respiration (Figs. 4, 5, Suppl. Figures 1–3) were compared using one-way analysis of variance (ANOVA; Prism 5.03; GraphPad Software, La Jolla, CA, USA) with Newman–Keuls post-test for multiple pair-wise comparisons. To compare means between two groups we used unpaired Student’s t-test (Fig. 3). P-values < 0.05 were considered to be statistically significant. All data were normally distributed, and no outliers were detected.
This is an exploratory study using mitochondrial parameters and levels of energy metabolites as major outcome variables. The experimenter was blinded to the animal groups during the measurements of corner and chimney tests. Apart from that, no blinding was performed in this study.