Legal Issues, Statistical Planning, and Randomization
Experiments were approved by local government authorities (Bezirksregierung Düsseldorf) in accordance with E.U. guidelines (Directive 2010/63/EU) for the care and use of laboratory animals. Sample size calculations determined that 12 animals per group were required for neurological examinations and histochemical studies, given that the effect size was 1.167, the alpha error was 5%, and the beta error (1 statistical power) was 20%. Experimenters were blinded by a third person not involved in the assessments randomizing the animals, weighing, and providing the food pellets.
Food Modifications and Murinometrics
Adult male C57BL6/j mice (8 weeks, 26–30 g; Harlan-Netherlands, Rossdorf, Germany) were randomized to three diets: (a) normal nutrition (C1000; 3518 kcal/kg, 20% protein (i.e., casein); Altromin, Lage, Germany), (b) energy-reduced nutrition (C1012 mod.; 1313 kcal/kg, 20% protein (casein); Altromin), and (c) protein-energy-reduced nutrition (C1003 mod.; 1300 kcal/kg, 8% protein (casein); Altromin). Diets were offered ad libitum over 7, 14, or 30 days. Animals were then submitted to 30 min intraluminal MCAO. Throughout the study, animals were housed in single cages in a 12 h:12 h light/dark cycle. Food consumption and calorie intake were measured daily. Body (i.e., nose-anus) length was determined prior to diet modification. Body weight and BMI were determined weekly. Stool changes and behavioral abnormalities, namely, spontaneous motor hypoactivity, were checked daily.
Mice were anesthetized with 1.0–1.5% isoflurane (30% O2, remainder N2O). Rectal temperature was maintained between 36.5 and 37.0 °C using a feedback-controlled heating system. Cerebral laser Doppler flow (LDF) was recorded using a flexible probe (Perimed, Järfälla, Sweden) attached to the skull overlying the core of the middle cerebral artery territory. A midline neck incision was made. The left common and external carotid arteries were isolated and ligated, and the internal carotid artery was temporarily clipped. A silicon-coated nylon monofilament (0.21-mm tip diameter; Doccol, Sharon, MA, USA) was introduced through a small incision of the common carotid artery and advanced to the circle of Willis for MCAO [5, 6]. Reperfusion was initiated by monofilament removal after 30 min. Thirty minutes of MCAO was chosen, since this model induced reproducible injury of the striatum and the most lateral cortex with little animal dropouts. After surgery, wounds were carefully sutured and anesthesia was discontinued. Twenty-four hours later, animals were evaluated using the Clark score , which captures general and focal neurological deficits. Immediately before sacrifice, plasma samples were obtained by cardiac puncture after 5 h fasting that were used for analysis of total cholesterol, low-density lipoprotein cholesterol (LDL), triglycerides, and glucose levels (ADVIA® 2400; Siemens, Erlangen, Germany). One set of animals (n = 12/group) was transcardially perfused with normal saline followed by 4% paraformaldehyde. The animals’ brains were cut into 20-μm-thick coronal sections for histochemical studies. Another set of animals (n = 6/group) was transcardially perfused with normal saline. From the animals’ brains, tissue samples were collected from the middle cerebral artery territory for Western blots and real-time quantitative polymerase chain reaction (qPCR) studies. For this purpose, a 2-mm-thick coronal brain slice ranging from 1 mm rostral to 1 mm caudal to the bregma was prepared, from which a triangular slice containing the striatum and the most lateral parietal cortex was dissected. This selection strategy was chosen to exclude partial volume effects of infarct reductions or expansions on gene expression results. From the same animals, liver samples were also obtained.
Coronal sections collected at millimeter intervals across the brain were stained with cresyl violet. Infarct volume was determined by subtracting the area of healthy tissue in the ischemic hemisphere from that in the contralesional hemisphere [5, 6].
Immunohistochemistry of IgG Extravasation
Brain sections obtained from the rostrocaudal level of the midstriatum were rinsed for 20 min in 0.3% H2O2 in 70% methanol/0.1 M phosphate-buffered saline (PBS), immersed in 0.1 M PBS containing 5% bovine serum albumin (BSA) (05470; Sigma-Aldrich, Darmstadt, Germany), and incubated for 1 h in biotinylated anti-mouse IgG (1:100; Santa Cruz, Heidelberg, Germany), followed by diaminobenzidine (DAB) tetrahydrochloride (D5905; Sigma-Aldrich) staining with an avidin-biotin complex peroxidase kit (Vectastain Elite; Vector Labs, Burlingame, CA, USA) . IgG extravasation was analyzed by measuring the area of IgG leakage.
Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick End Labeling
Adjacent brain sections were subjected to terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) using a commercially available In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) [5, 6]. TUNEL+, that is, DNA-fragmented cells were evaluated under a Zeiss AxioObserver.Z1 microscope equipped with Apotome optical sectioning by counting the total number of labeled cells in the striatum.
Immunohistochemistry for Neuronal, Microglial, Astrocytic, and Inflammation Markers
Adjacent sections were immersed in 0.1 M PBS containing 0.3% Triton X-100 (PBS-T) and 5% normal donkey serum (D9663; Sigma-Aldrich). Sections were incubated overnight at 4 °C in monoclonal rabbit anti-NeuN (1:400; ab177487; Abcam, Cambridge, UK), monoclonal rat anti-CD45 (1:200; 550539; BD Biosciences, Heidelberg, Germany), polyclonal rabbit anti-ionized calcium binding adaptor protein (Iba)-1 (1:500; Wako Chemicals, Neuss, Germany), monoclonal rat anti-glial fibrillary acidic protein (GFAP) (1:200; 130300; Invitrogen, Dublin, Ireland), or polyclonal rabbit anti-inducible nitric oxide synthase (iNOS) (1:100; sc-650; Santa Cruz, CA, USA) antibodies that were detected with Alexa Fluor-488– or Alexa Fluor-594–labeled secondary antibodies (NeuN, Iba-1, GFAP, and iNOS) or biotinylated secondary antibodies followed by DAB staining with an avidin-biotin complex peroxidase kit (Vectastain Elite, Burlingame, CA, USA) (CD45). NeuN, Iba-1, GFAP, and iNOS labelings were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (D9542; Sigma-Aldrich). Sections were evaluated under a motorized Zeiss AxioObserver.Z1 inverted epifluorescence microscope equipped with Apotome optical sectioning (NeuN, Iba-1, GFAP, and iNOS) or an Olympus X52 microscope (CD45) by counting the total number of NeuN+, CD45+, or iNOS+ cells in the striatum, in which ischemic injury is most reproducible, or analyzing the area covered by activated microglia (Iba-1) or reactive astrocytes (GFAP). The latter analysis was preferred to cell counting, since individual cells could not always unequivocally be discriminated. The latter data were shown as percent changes.
Real-Time Quantitative Polymerase Chain Reaction
From the brain and liver tissue samples, messenger RNA (mRNA) was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). mRNA was converted to cDNA using a high-capacity RNA-to-cDNA kit (Thermo Fisher Scientific, Langenselbold, Germany). Real-time qPCR was performed in a StepOnePlus real-time PCR system using primers selected by the PubMed primer-BLAST tool (https://blast.ncbi.nlm.nih.gov/) (Suppl. Table 1). The efficiency of these primers had been confirmed in melting curves. β-Glucuronidase (β-Gluc) was used as a housekeeping gene; the brain and liver tissue from healthy mice served as control. Results were quantified using the 2−∆∆Ct method. PCR were performed in triplicate, of which mean values were formed for each mouse.
During the mRNA extraction, protein samples were collected after bromochloropropane (B9673; Sigma-Aldrich) separation. Ethanol was added and samples centrifuged at 12.000g for 5 min. This procedure was repeated twice. The resulting pellet was suspended in 4% sodium dodecyl sulfate (SDS) (436143; Sigma-Aldrich). Protein content was measured using the Bradford method. Equal amounts of protein (20 μg) were loaded on 10% SDS-polyacrylamide gels, submitted to SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA). Membranes were blocked by 5% non-fat-dried milk (M7409; Sigma-Aldrich) in 50 mM Tris-buffered saline (TBS) containing 0.1% Tween (P9416; Sigma-Aldrich) for 1 h at room temperature, washed, and incubated overnight at 4 °C with monoclonal rabbit anti-Sirt-1 (1:2000; ab32441; Abcam) and polyclonal rabbit anti-β-actin (1:10000; 4967; Cell Signaling, Frankfurt, Germany) antibodies. The next day, membranes were washed and incubated with secondary donkey anti-rabbit antibody. Blots were revealed using a chemiluminescence kit and scanned using a myECL Imager (Thermo Fisher Scientific). Sirt-1 abundance was densitometrically evaluated in three independent experiments. The relative abundance of Sirt-1 was normalized to protein loading as determined in β-actin blots.
Statistical analyses were performed using SPSS for Windows. Nutritional data and LDF recordings were analyzed by two-way repeated measurement ANOVA followed by unpaired t tests as post hoc tests. Neurological deficits and histochemical data were analyzed by one-way ANOVA followed by Tukey post hoc tests (for normally distributed results) or Kruskal-Wallis tests (for non-normally distributed results). Real-time qPCR data were compared by pairwise t tests. To explore the relationship of calorie intake with infarct volume and neurological deficits, two-tailed Pearson’s correlations were computed. Nutritional data, LDF recordings, and real-time qPCR data are presented as mean ± S.D. values. Neurological deficits, histochemical data, and Western blots are shown as median ± interquartile range box plots with minimum/maximum data as whiskers. p values < 0.05 were defined to indicate statistical significance.