Materials
10 mg of the inflammasome-inhibitor MCC950 (#538120, Merck, Darmstadt, Germany) were dissolved in 1 ml of sterile water and further diluted with PBS to the final concentration of 5.5 mg/ml or 50 mg/kg body weight (BW), respectively. PBS, sterile water, anti-NeuN (MAB377) and anti-β-Actin (A5441) were all purchased by Merck. TRIzol reagent (#15596026), TE buffer (#12090015), TaqMan Reverse Transcription Reagents (#N8080234), random hexamer primers (#SO142), the rtPCR primers for Caspase 1 (Csp1, Mm00438023_m1, #4331182), Interleukin 1β (Il1b, Mm00434228_m1. #4331182), Interleukin 18 (Il18, Mm00434225_m1, #4331182) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH; TaqMan Predeveloped Assay Reagents for gene expression, #4352339E), ProLong Gold Antifade Mountant with DAPI dye (#P36931) and as secondary antibodies Alexa FluorTM 488 goat anti-mouse IgG (A11001), 488 donkey anti-rabbit IgG (A21206), 647 goat anti-rat IgG (A21247), 546 goat anti-rabbit IgG (A11035) and 488 donkey anti-rat IgG (A21208,) were provided by Thermo Fisher Scientific (Waltham, MA, USA). Anti-NLRP3 (AG-20B-0014) was purchased by Adipogen Life Sciences (San Diego, CA, USA). Anti-CD4 (#100506), and anti-Ly6G (#127636) were obtained by BioLegend (San Diego, CA, USA). Anti-GFAP (ab7260), anti-MAP2 (ab32454) and anti-Iba1 (ab178846) were produced by Abcam (Cambridge, UK). Peroxidase AffiniPure Donkey Anti-Mouse IgG (#715–035-150) was delivered by Jackson ImmunoResearch (Cambridge, UK) (see also Additional file 1: Tables S1, S2).
Animals and sample size calculation
We used 6–8-week-old male C57Bl/6 N mice, purchased from Charles River Laboratories (Sulzfeld, Germany). In all, 53 mice were used for this study. 10 mice were assigned to a 24 h post-tMCAO treatment group (MCC950 d1), 9 mice were assigned to a prophylactic pre-tMCAO treatment group (MCC950 d0), and 10 mice to the respective vehicle groups each. Each of the aforementioned groups passed a 7 day long reperfusion phase. Since the two vehicle groups did not differ regarding mortality (Additional file 1: Fig. S1), infarct size and clinical tests, the surviving animals in the vehicle groups were pooled to reduce the total number of necessary animals best possibly according to the 3R-principle. Moreover, 14 mice were used as sham and vehicle groups for a post 24 h reperfusion study. After randomization, tMCAO was conducted for 30 min. Surgery and evaluation of all readout parameters were performed blinded to the experimental groups. Assuming a reduction of infarct volume of 30% as functionally relevant and a standard deviation of 20% to the respective mean values, a group size of ≥ 8 was necessary to show this effect with a power of 0.8 and a probability of a type I error of < 0.5 (calculated with GraphPad StatMate 2.00). All in all, 27 mice survived the 7 day observation period (11 in the pooled vehicle group, 8 in the MCC950 d0 group, 8 in the MCC950 d1 group) and were used for infarct size measurement and clinical tests. Out of these, 9 mice of the vehicle group, 7 of the MCC950 d0 group and 7 of the MCC950 d1 group were used for further immunohistological and Western Blot analysis. 14 mice (7 vehicle and 7 sham) were used for Western Blot analysis after only 24 h of reperfusion.
Animal treatment
Animals were treated with 100 μl of the inflammasome-inhibitor MCC950 (50 mg/kg BW) or the same volume of vehicle (1 × PBS), which were administered by an intraperitoneal injection 24 h after tMCAO (MCC950 d1 group) or directly before occluding the MCA for 30 min (MCC950 d0 group) [11].
Ischemia model
The tMCAO model was used to induce focal cerebral ischemia as described in detail before [16]. The experiments were carried out blinded. An independent researcher who was not involved in data analysis coded and randomized the mice. To reduce the variability of our outcome parameters caused by sex-differences only male mice were used in the study [17]. Before tMCAO the mice were anesthetized with 2% isoflurane. 200 mg/kg BW metamizol was injected subcutaneously and 4% lidocaine gel applied on the wound margins as analgesia. With a servo-controlled heating blanket, a body core temperature close to 37 °C was maintained throughout surgery. After a midline neck incision, a standardized silicon rubber-coated 6.0 nylon monofilament (6023910PK10; Doccol, Sharon, MA, USA) was inserted into the right common carotid artery and advanced via the internal carotid artery to occlude the origin of the MCA. After 30 min, the mice were re-anesthetized and the occluding filament removed to allow reperfusion. To reduce infarct variability all mice were operated by the same operator. Operation time did not exceed 15 min. The observation period accounted for 7 days. Sham mice were treated like mice of the vehicle group but the filament was removed directly after its insertion.
Stroke volume
Animals were killed 7 days after tMCAO and the brains were cut in three 2 mm-thick coronal sections. The slices were stained for 20 min at 37 °C with 2% TTC to visualize the infarctions. Edema-corrected infarct volumes were calculated by planimetry (ImageJ software, National Institutes of Health, Bethesda, MD, USA) [18]. Moreover, one additional 10 µm thick slice, obtained out of the middle coronal section, was also analyzed histologically by MAP2 staining to depict neuronal damage as described previously [19].
Assessment of functional outcome
The global neurological deficits were quantified conducting the Neuro Score, composed of the sum of the inverted Bederson score as well as the grip test. They were assessed after anesthesia abated at the day of the tMCAO experiments (day 0) as well as every 24 h until day 7 after stroke induction [20, 21]. Furthermore, the latency to move test, which measures the time a mouse needs to move one body length, and the modified Neurological Severity Scores (mNSS), a composite of motor, sensory, reflex and balance tests resulting in a deficit score from 0 (normal function) to 18 (maximal deficit), were assessed at the same timepoints [22, 23]. The clinical assessment at days 1–24 h post-tMCAO was performed directly before the treatment in the MCC950 d1 group took place.
Exclusion criteria
Mice were excluded from endpoint analyses due to: (1) death before the predefined experimental endpoint; (2) Bederson score = 5 (at any timepoint after tMCAO); (3) weight loss > 20% at any timepoint after tMCAO. All in all, 3 mice were excluded due to death during the surgery and 2 due to weight loss > 20%.
Quantitative real-time polymerase chain reaction (PCR)
After sacrification of the animals, we separated the cortices and basal ganglia tissue from both hemispheres for RNA isolation. Next to homogenization with TRIzol Reagent (1 ml per 100 mg tissue), chlorophorm was added and samples were centrifuged at 12,000 g for 5 min at 4 °C. The upper aqueous phase was collected and mixed with isopropyl alcohol for RNA precipitation, washed, dissolved in TE buffer and finally quantified spectrophotometrically. 1 µg of total RNA were used for reverse transcription with the TaqMan Reverse Transcription Reagents according to the manufacturer’s protocol using random hexamers. Primers for IL1β, IL18 and Caspase 1 were utilized. GAPDH was used as endogenous control. PCR was performed with equal amounts of cyclic deoxyribonucleic acid and water control in the StepOnePlus™ Real-Time PCR System (Applied Biosystems) using the TaqMan Universal 2X PCR Master Mix (Applied Biosystems). Each sample was measured in duplicate and all data points examined for integrity by analysis of the amplification plot. The comparative cycle threshold method was used for relative quantification of gene expression as described elsewhere [24].
Protein extraction and Western Blot analysis
Protein extraction and Western blot analysis were performed according to standard procedures as previously described [25]. Therefore, dissected cortices and basal ganglia from the mouse brains were homogenized with RIPA buffer (25 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS) containing 4% proteinase inhibitor (cOmpleteTM protease inhibitor cocktail, Thermo Fisher Scientific) and sonified for 10 s. After centrifugation at 15,000 g for 30 min at 4 °C supernatants were used for bicinchoninic acid protein assay and subsequent Western Blot analysis. The lysates were mixed with 2 × SDS–PAGE loading buffer (final concentration: 60 mM Tris pH 6.8, 10% beta-mercaptoethanol, 5% SDS, 10% glycerol) at 95 °C for 10 min. 20 µg of total protein was loaded on the gel, electrophoresed and transferred to a nitrocellulose membrane. After blocking for 30 min with blocking buffer (5% nonfat dry milk, PBS, 0.05% Tween-20) membranes were incubated with the primary antibodies anti-GFAP (1:10.000), anti-NLRP3 (1:500), anti-Iba1 (1:500) and anti-actin mAb (1:1.000.000) at 4 °C overnight. As secondary antibodies Peroxidase AffiniPure Donkey Anti-Mouse IgG or Goat Anti-Rabbit IgG (1:2000) were used.
Histology and immunohistochemistry
For histology brain tissue was cut in 2-mm-thick coronal sections, embedded in Tissue-Tek OCT compound and frozen. Brain sections were cut on a cryostat into 10-μm thin slices and used for all analysis. For immunohistochemistry, slices were post-fixated with ice-cold 100% methanol. Immunohistochemistry was performed according to standard procedures [26]. Mouse brains were stained for DAPI and with anti-GFAP (1:100), anti-NeuN (1:100), anti-CD4 (1:100), anti-Iba1 (1:100), anti-Ly.6 g (1:50) and anti-NLRP3 (1:100). Secondary antibodies were used in a dilution of 1:100. For measurement of the NeuN intensity, images at the level of the basal ganglia (0.4, 0.45 and 0.5 mm anterior from bregma) of 7–9 different animals for each group were recorded with a microscope (Leica DMi8 equipped with the DMC 2900/DFC 3000G camera control and LAS X software (Leica, Wetzlar, Germany)). Subsequently, after converting the images into 16-bit black and white files, the intensity of the NeuN staining was compared between the ipsilesional and contralesional hemispheres with ImageJ Analysis Software 1.52a (National Institutes of Health, Bethesda, MD, USA). For this purpose, the ratio of the overall intensity scores of the ipsilesional and contralesional hemisphere was calculated and served as a comparison parameter among each single animal. Within the same three brain sections per animal, NeuN counting was performed directly under the microscope in all together 10 priorly defined brain regions as outlined in the respective figure. The countings of CD4, Iba1 and Ly.6 g positive cells were performed within 5 ipsilesional and 5 contralesional priorly defined specific segments (in each case 3 cortical and 2 basal ganglial segments). The recording took place with the aforementioned microscope. The cells within the segments were counted manually.
Statistical analysis
The results of the neurological tests are presented as bar graphs with the mean and the standard error of the mean (SEM) (latency to move test) or inter quartile range (IQR) (Neuro Score and mNSS). All the other results are presented as box plots indicating median, the 25th percentile, the 75th percentile, minimum and maximum. For statistical analysis the GraphPad Prism 8 software was used. Data were tested for Gaussian distribution with the D’Agostino-Pearson omnibus normality test and then analyzed by one-way analysis of variance (ANOVA) and in the case of repeated measurements two-way ANOVA with post hoc Tukey adjustment for p values or unpaired t test. Probability values < 0.05 were considered to indicate statistically significant results. A Log-rank (Mantel–Cox) test was used for the survival curves.